Fgfr1 extracellular domain combination therapies

ABSTRACT

Methods of treating cancer comprising administering a fibroblast growth factor receptor 1 (FGFR1) extracellular domain (ECD) and/or an FGFR1 ECD fusion molecule in combination with at least one additional therapeutic agent selected from docetaxel, paclitaxel, vincristine, carboplatin, cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU), leucovorin, pemetrexed, and bevacizumab are provided. Dosage packs comprising an FGFR1 ECD and/or an FGFR1 ECD fusion molecule and/or at least one additional therapeutic agent selected from docetaxel, paclitaxel, vincristine, carboplatin, cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU), leucovorin, pemetrexed, and bevacizumab are also provided. In some embodiments, a dosage pack comprises instructions for administering FGFR1 ECD and/or FGFR1 ECD fusion molecule with at least one additional therapeutic agent.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/577,330, filed Dec. 19, 2014, which is a continuation of U.S. patentapplication Ser. No. 13/296,168, filed Nov. 14, 2011, now U.S. Pat. No.8,951,972, issued Feb. 10, 2015, which claims priority to U.S.Provisional Application No. 61/421,462 filed Dec. 9, 2010, the contentsof each of which is incorporated herein by reference in its entirety forany purpose.

BACKGROUND AND SUMMARY OF THE INVENTION

Soluble forms of Fibroblast Growth Factor Receptor 1 (FGFR1) have beenshown to inhibit tumor cell growth in vitro and in vivo. See, e.g., U.S.Pat. No. 7,678,890. Combining an anti-cancer therapeutic molecule, suchas a soluble form of FGFR1, with another anti-cancer therapeuticmolecule, can result in antagonistic, additive, or synergistic effectson the efficacy of each of the anti-cancer therapeutics.

The inventors have discovered that administration of an FGFR1 ECD anddocetaxel in a mouse xenograft model of non-small cell lung cancer showssynergistic anti-tumor activity of the therapeutic agents. In addition,the inventors have discovered that administration of an FGFR1 ECD and atleast one additional therapeutic molecule selected from paclitaxel,vincristine, carboplatin, cisplatin, oxaliplatin, doxorubicin,5-fluorouracil (5-FU), leucovorin, pemetrexed, and bevacizumab, has atleast additive activity relative to each molecule administered alone incertain mouse xenograft models.

In some embodiments, methods of treating cancer comprising administeringto a subject a fibroblast growth factor receptor 1 (FGFR1) extracellulardomain (ECD) and at least one additional therapeutic agent selected fromdocetaxel, paclitaxel, vincristine, carboplatin, cisplatin, oxaliplatin,doxorubicin, 5-fluorouracil (5-FU), leucovorin, pemetrexed, sorafenib,etoposide, topotecan, a vascular endothelial growth factor (VEGF)antagonist, a VEGF trap, an anti-VEGF antibody, and bevacizumab areprovided. In some embodiments, the at least one additional therapeuticagent is docetaxel. In some embodiments, the at least one additionaltherapeutic agent is pemetrexed. In some embodiments, the at least oneadditional therapeutic agent is cisplatin. In some embodiments, the atleast one additional therapeutic agent is paclitaxel. In someembodiments, the at least one additional therapeutic agent is 5-FU. Insome embodiments, the at least one additional therapeutic agent istopotecan. In some embodiments, the at least one additional therapeuticagent is viscristine. In some embodiments, the at least one additionaltherapeutic agents is a VEGF antagonist, such as an anti-VEGF antibodyor a VEGF trap. In some embodiments, the at least one additionaltherapeutic agent is bevacizumab. In some embodiments, the at least oneadditional therapeutic agent is sorafenib. In some embodiments, theFGFR1 ECD comprises a sequence selected from SEQ ID NOs: 1 to 4.

In some embodiments, methods of treating cancer comprising administeringto a subject a fibroblast growth factor receptor 1 (FGFR1) extracellulardomain (ECD) fusion molecule and at least one additional therapeuticagent selected from docetaxel, paclitaxel, vincristine, carboplatin,cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU), leucovorin,pemetrexed, sorafenib, etoposide, topotecan, a vascular epithelialgrowth factor (VEGF) antagonist, a VEGF trap, an anti-VEGF antibody, andbevacizumab are provided, wherein the FGFR1 ECD fusion moleculecomprises an FGFR1 ECD and a fusion partner. In some embodiments, the atleast one additional therapeutic agent is docetaxel. In someembodiments, the at least one additional therapeutic agent ispemetrexed. In some embodiments, the at least one additional therapeuticagent is cisplatin. In some embodiments, the at least one additionaltherapeutic agent is paclitaxel. In some embodiments, the at least oneadditional therapeutic agent is 5-FU. In some embodiments, the at leastone additional therapeutic agent is viscristine. In some embodiments,the at least one additional therapeutic agent is topotecan. In someembodiments, the at least one additional therapeutic agent is a VEGFantagonist, such as an anti-VEGF antibody or a VEGF trap. In someembodiments, the at least one additional therapeutic agent isbevacizumab. In some embodiments, the at least one additionaltherapeutic agent is sorafenib.

In some embodiments, methods of treating cancer comprising administeringto a subject an FGFR1 ECD or FGFR1 ECD fusion molecule and at least twoadditional therapeutic agents selected from docetaxel, paclitaxel,vincristine, carboplatin, cisplatin, oxaliplatin, doxorubicin,5-fluorouracil (5-FU), leucovorin, pemetrexed, etoposide, sorafenib,etoposide, topotecan, a vascular epithelial growth factor (VEGF)antagonist, a VEGF trap, an anti-VEGF antibody, and bevacizumab areprovided, wherein the FGFR1 ECD fusion molecule comprises an FGFR1 ECDand a fusion partner. In some embodiments, at least one of the twoadditional therapeutic agents is paclitaxel. In some embodiments, atleast one of the two additional therapeutic agents is cisplatin. In someembodiments, at least one of the two additional therapeutic agents iscarboplatin. In some embodiments, at least one of the two additionaltherapeutic agents is oxaliplatin. In some embodiments, at least one ofthe two additional therapeutic agents is 5-FU. In some embodiments, atleast one of the two additional therapeutic agents is doxorubicin. Insome embodiments, at least one of the two additional therapeutic agentsis etoposide. In some embodiments, at least one of the two additionaltherapeutic agents is topotecan. In some embodiments, at least one ofthe two additional therapeutic agents is a VEGF antagonist, such as ananti-VEGF antibody or a VEGF trap. In some embodiments, at least one ofthe two additional therapeutic agents is bevacizumab. In someembodiments, the two additional therapeutic agents are paclitaxel andcarboplatin. In some embodiments, the two additional therapeutic agentsare doxorubicin and paclitaxel. In some embodiments, the two additionaltherapeutic agents are cisplatin and etoposide. In some embodiments, thetwo additional therapeutic agents are oxaliplatin and 5-FU. In someembodiments, the two additional therapeutic agents are 5-FU andleucovorin. In some embodiments, the two additional therapeutic agentsare 5-FU and bevacizumab. In some embodiments, the two additionaltherapeutic agents are paclitaxel and bevacizumab. In some embodiments,the two additional therapeutic agents are cisplatin and etoposide.

In some embodiments, methods of treating cancer comprising administeringto a subject an FGFR1 ECD or FGFR1 ECD fusion molecule and at leastthree additional therapeutic agents selected from docetaxel, paclitaxel,vincristine, carboplatin, cisplatin, oxaliplatin, doxorubicin,5-fluorouracil (5-FU), leucovorin, pemetrexed, etoposide, sorafenib, aVEGF antagonist, an anti-VEGF antibody, a VEGF trap, and bevacizumab areprovided, wherein the FGFR1 ECD fusion molecule comprises an FGFR1 ECDand a fusion partner. In some embodiments, at least one of the threeadditional therapeutic agents is oxaliplatin. In some embodiments, atleast one of the three additional therapeutic agents is 5-FU. In someembodiments, at least one of the three additional therapeutic agents isleucovorin. In some embodiments, at least one of the three additionaltherapeutic agents is carboplatin. In some embodiments, at least one ofthe three additional therapeutic agents is paclitaxel. In someembodiments, at least one of the three additional therapeutic agents isbevacizumab. In some embodiments, the three additional therapeuticagents are oxaliplatin, 5-FU and leucovorin. In some embodiments, thethree additional therapeutic agents are bevacizumab, 5-FU andleucovorin. In some embodiments, at least two of the three additionaltherapeutic agents are cisplatin and etoposide.

The invention also relates, in some embodiments, to a combination of anFGFR1 ECD or FGFR1 ECD fusion molecule and at least one additionaltherapeutic agent selected from docetaxel, paclitaxel, vincristine,carboplatin, cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU),leucovorin, pemetrexed, sorafenib, etoposide, topotecan, a vascularendothelial growth factor (VEGF) antagonist, a VEGF trap, an anti-VEGFantibody, and bevacizumab, for treatment of cancer. The inventionfurther relates, in some embodiments to a combination of an FGFR1 ECD orFGFR1 ECD fusion molecule and at least two additional therapeutic agentsselected from docetaxel, paclitaxel, vincristine, carboplatin,cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU), leucovorin,pemetrexed, etoposide, sorafenib, etoposide, topotecan, a vascularepithelial growth factor (VEGF) antagonist, a VEGF trap, an anti-VEGFantibody, and bevacizumab, for treatment of cancer. The invention alsorelates, in some embodiments, to a combination of an FGFR1 ECD or FGFR1ECD fusion molecule and at least three additional therapeutic agentsselected from docetaxel, paclitaxel, vincristine, carboplatin,cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU), leucovorin,pemetrexed, etoposide, sorafenib, a VEGF antagonist, an anti-VEGFantibody, a VEGF trap, and bevacizumab, for treatment of cancer.

In some embodiments, the FGFR1 ECD and/or FGFR1 ECD fusion molecule ispackaged separately from the at least one, at least two, or at leastthree additional therapeutic agents. In some embodiments, the FGFR1 ECDand/or FGFR1 ECD fusion molecule is not mixed with the at least one, atleast two, or at least three additional therapeutic agents prior toadministration.

In some embodiments, a method of treating cancer comprisingadministering to a subject an FGFR1-ECD.339-Fc and docetaxel isprovided, wherein the FGFR1-ECD.339-Fc comprises the amino acid sequenceof SEQ ID NO: 6. In some embodiments, a method of treating cancercomprising administering to a subject an FGFR1-ECD.339-Fc and docetaxelis provided, wherein the FGFR1-ECD.339-Fc consists of the amino acidsequence of SEQ ID NO: 6.

In some embodiments, an FGFR1 ECD or FGFR1 ECD fusion molecule isglycosylated and/or sialylated. In some embodiments, an FGFR1 ECD or thepolypeptide portion of the FGFR1 ECD fusion molecule is expressed inChinese hamster ovary (CHO) cells. In some embodiments, an FGFR1 ECDcomprises an amino acid sequence selected from SEQ ID NO: 1 and SEQ IDNO: 3.

In some embodiments, at least one dose of an FGFR1 ECD and/or an FGFR1ECD fusion molecule and at least one dose of at least one additionaltherapeutic agent are administered concurrently. In some embodiments, atleast one dose of an FGFR1 ECD and/or an FGFR1 ECD fusion molecule andat least one dose of at least one additional therapeutic agent areadministered at the same time.

In some embodiments, the FGFR1 ECD comprises an amino acid sequenceselected from SEQ ID NOs: 1 to 4. In some embodiments, at least onefusion partner is selected from an Fc, albumin, and polyethylene glycol.In some embodiments, at least one fusion partner is an Fc. In someembodiments, the Fc comprises an amino acid sequence selected from SEQID NOs: 8 to 10. In some embodiments, the FGFR1 ECD fusion moleculecomprises a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 6. Insome embodiments, the at least one fusion partner is an Fc andpolyethylene glycol. In some embodiments, the at least one fusionpartners is polyethylene glycol. In some embodiments, the fusionmolecule comprises a linker between the FGFR1 ECD and one or more fusionpartners. In some embodiments, the FGFR1 ECD comprises a signal peptide.In some embodiments, the signal peptide comprises the amino acidsequence of SEQ ID NO: 7.

In some embodiments, the FGFR1 ECD fusion molecule is an amount in therange of about 0.5 mg/kg body weight to about 20 mg/kg body weight, suchas an amount in the range of about 8 to about 16 mg/kg body weight. Insome embodiments, the therapeutically effective amount of the FGFR1 ECDfusion molecule is a dose of about 8 mg/kg body weight, while in someembodiments, the therapeutically effective amount of the FGFR1 ECDfusion molecule is a dose of about 16 mg/kg body weight (or at about 10mg/kg body weight or about 20 mg/kg body weight, respectively, whencalculated using an extinction coefficient of 1.11 mL/mg*cm). In someembodiments, the therapeutically effective amount of FGFR1 ECD fusionmolecule is a dose of about 20 mg/kg body weight. In some embodiments,dosages may be administered twice a week, weekly, every other week, at afrequency between weekly and every other week, every three weeks, everyfour weeks, or every month.

The invention also relates, in some embodiments, to a dosage pack. Insome embodiments, a dosage pack comprising at least one componentselected from: (i) a fibroblast growth factor receptor 1 (FGFR1)extracellular domain (ECD), (ii) a fibroblast growth factor receptor 1(FGFR1) extracellular domain (ECD) fusion molecule, and (iii) at leastone additional therapeutic agent selected from docetaxel, paclitaxel,vincristine, carboplatin, cisplatin, oxaliplatin, doxorubicin,5-fluorouracil (5-FU), leucovorin, pemetrexed, etoposide, topotecan, aVEGF antagonist, an anti-VEGF antibody, a VEGF trap, bevacizumab, andsorafenib; and instructions for administering an FGFR1 ECD or FGFR1 ECDfusion molecule and the at least one additional therapeutic to a patientis provided. In some embodiments, the instructions included with thedosage pack comprise instructions for administering a therapeuticallyeffective amount of FGFR1 ECD fusion molecule. In some embodiments, thetherapeutically effective amount of the FGFR1 ECD fusion molecule is anamount in the range of about 0.5 mg/kg body weight to about 20 mg/kgbody weight, such as an amount in the range of about 8 to about 16 mg/kgbody weight. In some embodiments, the therapeutically effective amountof the FGFR1 ECD fusion molecule is a dose of about 8 mg/kg body weight.In some embodiments, the therapeutically effective amount of the FGFR1ECD fusion molecule is a dose of about 16 mg/kg body weight. In someembodiments, the therapeutically effective amount of FGFR1 ECD fusionmolecule is a dose of about 20 mg/kg body weight. In some embodiments,dosages may be administered twice a week, weekly, every other week, at afrequency between weekly and every other week, every three weeks, everyfour weeks, or every month.

In some embodiments, the dosage pack comprises an FGFR1 ECD and does notcomprise the at least one additional therapeutic agent, e.g., docetaxel,paclitaxel, vincristine, carboplatin, cisplatin, oxaliplatin,doxorubicin, 5-fluorouracil (5-FU), leucovorin, pemetrexed, etoposide,topotecan, VEGF antagonist, anti-VEGF antibody, VEGF trap, bevacizumab,or sorafenib. In some embodiments, the dosage pack comprises an FGFR1ECD fusion molecule and does not comprise the at least one additionaltherapeutic agent, e.g., docetaxel, paclitaxel, vincristine,carboplatin, cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU),leucovorin, pemetrexed, etoposide, topotecan, VEGF antagonist, anti-VEGFantibody, VEGF trap, bevacizumab, or sorafenib. In some embodiments, thedosage pack comprises at least one additional therapeutic agent, butdoes not comprise an FGFR1 ECD or an FGFR1 ECD fusion molecule. In someembodiments, the dosage pack comprises: (i) an FGFR1 ECD or an FGFR1 ECDfusion molecule, and (ii) at least one additional therapeutic agent. Insome embodiments, the at least one additional therapeutic agent isdocetaxel, paclitaxel, vincristine, carboplatin, cisplatin, oxaliplatin,doxorubicin, 5-fluorouracil (5-FU), leucovorin, pemetrexed, etoposide,topotecan, a VEGF antagonist, an anti-VEGF antibody, a VEGF trap,bevacizumab, or sorafenib. In some embodiments, the at least oneadditional therapeutic agent is docetaxel. In some embodiments, the atleast one additional therapeutic agent is pemetrexed. In someembodiments, the at least one additional therapeutic agent is cisplatin.In some embodiments, the at least one additional therapeutic agent ispaclitaxel. In some embodiments, the at least one additional therapeuticagent is 5-FU. In some embodiments, the at least one additionaltherapeutic agent is viscristine. In some embodiments, the at least oneadditional therapeutic agent is bevacizumab. In some embodiments, the atleast one additional therapeutic agent is sorafenib. In someembodiments, the dosage pack comprises at least two additionaltherapeutic agents, but does not comprise an FGFR1 ECD or an FGFR1 ECDfusion molecule. In some embodiments, the at least two additionaltherapeutic agents are chosen from docetaxel, paclitaxel, vincristine,carboplatin, cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU),leucovorin, pemetrexed, etoposide, topotecan, a VEGF antagonist, ananti-VEGF antibody, a VEGF trap, bevacizumab, and sorafenib.

In some embodiments described above, the FGFR1 ECD or the FGFR1 ECDportion of the FGFR1 ECD fusion molecule comprises a sequence selectedfrom SEQ ID NOs: 1 to 4. In some embodiments, at least one fusionpartner is selected from an Fc, albumin, and polyethylene glycol. Insome embodiments, at least one fusion partner is an Fc. In someembodiments, the Fc comprises an amino acid sequence selected from SEQID NOs: 8 to 10. In some embodiments, the FGFR1 ECD fusion moleculecomprises a sequence selected from SEQ ID NO: 5 and SEQ ID NO: 6. Insome embodiments, the FGFR1 ECD fusion molecule consists of a sequenceselected from SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, the atleast one fusion partner is an Fc and polyethylene glycol. In someembodiments, the at least one fusion partners is polyethylene glycol.

In certain embodiments, the cancer is prostate cancer, breast cancer,colorectal cancer, lung cancer, endometrial cancer, head and neckcancer, laryngeal cancer, liver cancer, renal cancer glioblastoma orpancreatic cancer. In certain embodiments, the cancer is lung cancer. Incertain embodiments, the cancer is renal cancer. In certain embodiments,the cancer is colon cancer. In certain embodiments, the cancer is breastcancer. In certain embodiments, the cancer is endometrial cancer. Incertain embodiments, the cancer is prostate cancer.

Any embodiment described herein or any combination of additionaltherapeutic agents thereof applies to any and all methods of theinvention described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the mean tumor volume in mice administered FGFR1-ECD.339-Fcalone, docetaxel alone, FGFR1-ECD.339-Fc and docetaxel sequentially, andFGFR1-ECD.339-Fc and docetaxel concurrently, as described in Example 1.

FIGS. 2A-B show the mean tumor volume (A) and body weight (B) of miceadministered FGFR1-ECD.339-Fc alone, pemetrexed alone (62.5 mg/kg dose),or FGFR1-ECD.339-Fc and pemetrexed (62.5 mg/kg dose), as described inExample 2.

FIGS. 3A-B show the mean tumor volume (A) and body weight (B) of miceadministered FGFR1-ECD.339-Fc alone, pemetrexed alone (125 mg/kg dose),or FGFR1-ECD.339-Fc and pemetrexed (125 mg/kg dose), as described inExample 2.

FIGS. 4A-B show the mean tumor volume (A) and body weight (B) of miceadministered FGFR1-ECD.339-Fc alone, pemetrexed alone (250 mg/kg dose),or FGFR1-ECD.339-Fc and pemetrexed (250 mg/kg dose), as described inExample 2.

FIG. 5 shows the mean tumor volume in mice administered FGFR1-ECD.339-Fcalone, cisplatin alone, or the combination of FGFR1-ECD.339-Fc andcisplatin, as described in Example 3A.

FIG. 6 shows the mean tumor volume in mice administered FGFR1-ECD.339-Fcalone, paclitaxel alone, or the combination of FGFR1-ECD.339-Fc andpaclitaxel, as described in Example 3B.

FIG. 7 shows the mean tumor volume in mice administered FGFR1-ECD.339-Fcalone, 5-FU alone, or the combination of FGFR1-ECD.339-Fc and 5-FU, asdescribed in Example 3C.

FIGS. 8A-B show the mean tumor volume in mice administeredFGFR1-ECD.339-Fc alone, docetaxel alone, or the combination ofFGFR1-ECD.339-Fc and docetaxel, at two different dosages of docetaxel, 3mg/kg (A) and 10 mg/kg (B), as described in Example 3D.

FIGS. 9A-C show the mean tumor volume in mice administeredFGFR1-ECD.339-Fc alone, vincristine alone, or the combination ofFGFR1-ECD.339-Fc and vincristine, at two different dosages ofvincristine, 1 mg/kg beginning on day 1 (A) and 1.5 mg/kg beginning onday 19 (B), as described in Example 3E. The mean body weight of miceadministered 1.5 mg/kg beginning on day 19 is also shown (C).

FIG. 10 shows the mean tumor volume in mice administeredFGFR1-ECD.339-Fc alone, carboplatin alone, paclitaxel alone, thecombination of carboplatin and paclitaxel, and the combination ofFGFR1-ECD.339-Fc, carboplatin, and paclitaxel, as described in Example3F.

FIGS. 11A-E show the mean tumor volume in mice administeredFGFR1-ECD.339-Fc alone, the combination of 5-FU (10 mg/kg) andleucovorin (10 mg/kg), and the combination of FGFR1-ECD.339-Fc, 5-FU (10mg/kg), and leucovorin (10 mg/kg) (A); FGFR1-ECD.339-Fc alone, thecombination of 5-FU (20 mg/kg) and leucovorin (20 mg/kg), and thecombination of FGFR1-ECD.339-Fc, 5-FU (20 mg/kg), and leucovorin (20mg/kg) (B); FGFR1-ECD.339-Fc alone, the combination of 5-FU (30 mg/kg)and leucovorin (30 mg/kg), and the combination of FGFR1-ECD.339-Fc, 5-FU(30 mg/kg), and leucovorin (30 mg/kg) (C); FGFR1-ECD.339-Fc alone,bevacizumab alone, and the combination of FGFR1-ECD.339-Fc andbevacizumab (D); and FGFR1-ECD.339-Fc alone, the combination ofbevacizumab, 5-FU, and leucovorin, and the combination ofFGFR1-ECD.339-Fc, bevacizumab, 5-FU, and leucovorin (E), as described inExample 4A.

FIGS. 12A-C show the mean tumor volume in mice administeredFGFR1-ECD.339-Fc alone, oxaliplatin plus 5-FU and leucovorin (LV) alone,and the combination of FGFR1-ECD.339-Fc and oxaliplatin plus 5-FU/LV, atdifferent dosages of oxaliplatin, 5 mg/kg (A), 10 mg/kg (B), and 15mg/kg (C), as described in Example 4B.

FIG. 13 shows the mean tumor volume in mice administeredFGFR1-ECD.339-Fc alone, the combination of doxorubicin and paclitaxel,and the combination of FGFR1-ECD.339-Fc, doxorubicin, and paclitaxel, asdescribed in Example 5.

FIG. 14 shows the mean tumor volume in mice administeredFGFR1-ECD.339-Fc alone, a Kinase Insert Domain Receptor (KDR)-ECD fusionmolecule alone, and the combination of FGFR1-ECD.339-Fc and the KDR-ECDfusion molecule, as described in Example 6.

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Certain techniques used in connection with recombinant DNA,oligonucleotide synthesis, tissue culture and transformation (e.g.,electroporation, lipofection), enzymatic reactions, and purificationtechniques are known in the art. Many such techniques and procedures aredescribed, e.g., in Sambrook et al. Molecular Cloning: A LaboratoryManual (2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)), among other places. In addition, certaintechniques for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients arealso known in the art.

In this application, the use of “or” means “and/or” unless statedotherwise. In the context of a multiple dependent claim, the use of “or”refers back to more than one preceding independent or dependent claim inthe alternative only. Also, terms such as “element” or “component”encompass both elements and components comprising one unit and elementsand components that comprise more than one subunit unless specificallystated otherwise.

As used herein, all numbers are approximate, and may be varied toaccount for measurement error and the rounding of significant digits.The use of “about” before certain measured quantities includesvariations due to sample impurities, measurement error, human error, andstatistical variation, as well as the rounding of significant digits.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The terms “nucleic acid molecule” and “polynucleotide” may be usedinterchangeably, and refer to a polymer of nucleotides. Such polymers ofnucleotides may contain natural and/or non-natural nucleotides, andinclude, but are not limited to, DNA, RNA, and PNA. “Nucleic acidsequence” refers to the linear sequence of nucleotides that comprise thenucleic acid molecule or polynucleotide.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Such polymers of amino acid residues may contain natural ornon-natural amino acid residues, and include, but are not limited to,peptides, oligopeptides, dimers, trimers, and multimers of amino acidresidues. Both full-length proteins and fragments thereof areencompassed by the definition. The terms also include post-expressionmodifications of the polypeptide, for example, glycosylation,sialylation, acetylation, phosphorylation, and the like. Furthermore,for purposes of the present invention, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions, andsubstitutions (generally conservative in nature), to the nativesequence, as long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe proteins or errors due to PCR amplification. When a polypeptide“consists of” a particular amino acid sequence, it may still containpost-translational modifications, such as glycosylation and sialylation.

The term “FGFR1 extracellular domain” (“FGFR1 ECD”) includes full-lengthFGFR1 ECDs, FGFR1 ECD fragments, and FGFR1 ECD variants. As used herein,the term “FGFR1 ECD” refers to an FGFR1 polypeptide that lacks theintracellular and transmembrane domains, with or without a signalpeptide. In some embodiment, the FGFR1 ECD is a human full-length FGFR1ECD having an amino acid sequence selected from SEQ ID NOs: 1 and 2. Theterm “full-length FGFR1 ECD”, as used herein, refers to an FGFR1 ECDthat extends to the last amino acid of the extracellular domain, and mayor may not include an N-terminal signal peptide. As defined herein, thelast amino acid of the full-length FGFR1 ECD is at position 353. Thus, ahuman full-length FGFR1 ECD may consist of the amino acid sequencecorresponding to SEQ ID NO.: 2 (mature form) or to SEQ ID NO.: 1 (withthe signal peptide). As used herein, the term “FGFR1 ECD fragment”refers to an FGFR1 ECD having one or more residues deleted from the Nand/or C terminus of the full-length ECD and that retains the ability tobind to FGF-2. The FGFR1 ECD fragment may or may not include anN-terminal signal peptide. In some embodiments, the FGFR1 ECD fragmentis a human FGFR1 ECD fragment having an amino acid sequencecorresponding to SEQ ID NO.: 4 (mature form) or to SEQ ID NO.: 3 (withthe signal peptide).

As used herein, the term “FGFR1 ECD variants” refers to FGFR1 ECDs thatcontain amino acid additions, deletions, and substitutions and thatremain capable of binding to FGF-2. Such variants may be at least 90%,92%, 95%, 97%, 98%, or 99% identical to the parent FGFR1 ECD. The %identity of two polypeptides can be measured by a similarity scoredetermined by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program with the default settings for determiningsimilarity. Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2:482-489 (1981) to find thebest segment of similarity between two sequences. In some embodiments,an FGFR1 ECD variant is at least 95% identical to the sequence of SEQ IDNO: 4.

A polypeptide having an amino acid sequence at least, for example, 95%identical to a reference amino acid sequence of an FGFR1 ECD polypeptideis one in which the amino acid sequence of the polypeptide is identicalto the reference sequence except that the polypeptide sequence mayinclude up to five amino acid alterations per each 100 amino acids ofthe reference polypeptide. In other words, to obtain a polypeptidehaving an amino acid sequence at least 95% identical to a referenceamino acid sequence, up to 5% of the amino acid residues in thereference sequence may be deleted or substituted with another aminoacid, or a number of amino acids, up to 5% of the total amino acidresidues in the reference sequence, may be inserted into the referencesequence. These alterations of the reference sequence may occur at theN- or C-terminal positions of the reference amino acid sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence, or in one or morecontiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least70%, 80%, 90%, or 95% identical to, for instance, an amino acid sequenceor to a polypeptide sequence encoded by a nucleic acid sequence setforth in the Sequence Listing can be determined conventionally usingknown computer programs, such the Bestfit program. When using Bestfit orother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,that the percentage of identity is calculated over the full length ofthe reference amino acid sequence and that gaps in homology of up to 5%of the total number of amino acid residues in the reference sequence areallowed.

As used herein, the terms “hFGFR1-ECD.353” and “hFGFR1.353” may be usedinterchangeably to refer to the full-length human FGFR1 ECDcorresponding to SEQ ID NO: 1 (with signal peptide) or to SEQ ID NO: 2(without signal peptide; mature form).

As used herein, the terms “hFGFR1-ECD.339” and “hFGFR1.339” may be usedinterchangeably to refer to the human FGFR1 ECD corresponding to SEQ IDNO: 3 (with signal peptide) or to SEQ ID NO: 4 (without signal peptide;mature form).

Additional hFGFR1 ECDs are described, for example, in U.S. Pat. No.7,678,890, which is incorporated by reference herein in its entirety forany purpose.

The term “FGFR1 ECD fusion molecule” refers to a molecule comprising anFGFR1 ECD, and one or more “fusion partners.” In some embodiments, theFGFR1 ECD and the fusion partner are covalently linked (“fused”). If thefusion partner is also a polypeptide (“the fusion partner polypeptide”),the FGFR1 ECD and the fusion partner polypeptide may be part of acontinuous amino acid sequence, and the fusion partner polypeptide maybe linked to either the N terminus or the C terminus of the FGFR1 ECD.In such cases, the FGFR1 ECD and the fusion partner polypeptide may betranslated as a single polypeptide from a coding sequence that encodesboth the FGFR1 ECD and the fusion partner polypeptide (the “FGFR1 ECDfusion protein”). In some embodiments, the FGFR1 ECD and the fusionpartner are covalently linked through other means, such as, for example,a chemical linkage other than a peptide bond. Many known methods ofcovalently linking polypeptides to other molecules (for example, fusionpartners) may be used. In other embodiments, the FGFR1 ECD and thefusion partner may be fused through a “linker,” which is comprised of atleast one amino acid or chemical moiety.

In some embodiments, the FGFR1 ECD polypeptide and the fusion partnerare noncovalently linked. In some such embodiments, they may be linked,for example, using binding pairs. Exemplary binding pairs include, butare not limited to, biotin and avidin or streptavidin, an antibody andits antigen, etc.

Exemplary fusion partners include, but are not limited to, animmunoglobulin Fc domain, albumin, and polyethylene glycol. The aminoacid sequences of some exemplary Fc domains are shown in SEQ ID NOs: 8to 10. In some embodiments, an FGFR1 ECD fused to an Fc is referred toas an “hFGFR1 ECD-Fc.” In some embodiments, the Fc domain is selectedfrom an IgG1 Fc, an IgG2 Fc, an IgG3 Fc, and an IgG4 Fc.

As used herein, the terms “hFGFR1-ECD.339-Fc” and “hFGFR1.339-Fc” may beused interchangeably to refer to an amino acid sequence selected fromSEQ ID NO: 6 (without signal peptide, mature form) and SEQ ID NO: 5(with signal peptide). Nonlimiting exemplary cancers that may be treatedwith hFGFR1-ECD.339-Fc include, but are not limited to, non-small celllung cancer, colon cancer, breast cancer, gastric cancer, head and neckcancer, prostate cancer, endometrial cancer, sarcoma, small cell lungcancer, ovarian cancer, Kaposi's sarcoma, Hodgkin's disease, leukemia,non-Hodgkin's lymphoma, neuroblastoma (brain cancer), rhabdomyosarcoma,Wilms' tumor, acute lymphoblastic leukemia, acute lymphoblasticleukemia, bladder cancer, testicular cancer, lymphomas, germ celltumors, cancers of the colon and rectum, gastrointestinal cancers,thyroid cancer, multiple myeloma, pancreatic cancer, mesothelioma,malignant pleural mesothelioma, hematological/lymphatic cancers,malignant peritoneal mesothelioma, esophageal cancer, renal cellcarcinoma, glioblastoma multiforme, and liver cancer.

The term “signal peptide” refers to a sequence of amino acid residueslocated at the N terminus of a polypeptide that facilitates secretion ofa polypeptide from a mammalian cell. A signal peptide may be cleavedupon export of the polypeptide from the mammalian cell, forming a matureprotein. Signal peptides may be natural or synthetic, and they may beheterologous or homologous to the protein to which they are attached.Exemplary signal peptides include, but are not limited to, FGFR1 signalpeptides, such as, for example, the amino acid sequence of SEQ ID NO: 7.Exemplary signal peptides also include signal peptides from heterologousproteins. A “signal sequence” refers to a polynucleotide sequence thatencodes a signal peptide. In some embodiments, an FGFR1 ECD lacks asignal peptide. In some embodiments, an FGFR1 ECD includes at least onesignal peptide, which may be a native FGFR1 signal peptide or aheterologous signal peptide.

The term “vector” is used to describe a polynucleotide that may beengineered to contain a cloned polynucleotide or polynucleotides thatmay be propagated in a host cell. A vector may include one or more ofthe following elements: an origin of replication, one or more regulatorysequences (such as, for example, promoters and/or enhancers) thatregulate the expression of the polypeptide of interest, and/or one ormore selectable marker genes (such as, for example, antibioticresistance genes and genes that may be used in colorimetric assays,e.g., β-galactosidase). The term “expression vector” refers to a vectorthat is used to express a polypeptide of interest in a host cell.

A “host cell” refers to a cell that may be or has been a recipient of avector or isolated polynucleotide. Host cells may be prokaryotic cellsor eukaryotic cells. Exemplary eukaryotic cells include mammalian cells,such as primate or non-primate animal cells; fungal cells; plant cells;and insect cells. Exemplary mammalian cells include, but are not limitedto, 293 and CHO cells, and their derivatives, such as 293-6E and DG44cells, respectively.

The term “isolated” as used herein refers to a molecule that has beenseparated from at least some of the components with which it istypically found in nature. For example, a polypeptide is referred to as“isolated” when it is separated from at least some of the components ofthe cell in which it was produced. Where a polypeptide is secreted by acell after expression, physically separating the supernatant containingthe polypeptide from the cell that produced it is considered to be“isolating” the polypeptide. Similarly, a polynucleotide is referred toas “isolated” when it is not part of the larger polynucleotide (such as,for example, genomic DNA or mitochondrial DNA, in the case of a DNApolynucleotide) in which it is typically found in nature, or isseparated from at least some of the components of the cell in which itwas produced, e.g., in the case of an RNA polynucleotide. Thus, a DNApolynucleotide that is contained in a vector inside a host cell may bereferred to as “isolated” so long as that polynucleotide is not found inthat vector in nature.

The term “anti-neoplastic composition” refers to a composition useful intreating cancer comprising at least one active therapeutic agent, e.g.,an “anti-cancer agent.” Examples of therapeutic agents (anti-canceragents) include, but are limited to, e.g., chemotherapeutic agents,growth inhibitory agents, cytotoxic agents, agents used in radiationtherapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents,and other agents to treat cancer, such as anti-VEGF antibodies (e.g.,bevacizumab, AVASTIN®), anti-HER-2 antibodies (e.g., trastuzumab,HERCEPTIN®), anti-CD20 antibodies (e.g., rituximab, RITUXAN®), anepidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosinekinase inhibitor), HER1/EGFR inhibitors (e.g., erlotinib (TARCEVA®),platelet derived growth factor inhibitors (e.g., GLEEVEC® (ImatinibMesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines,antagonists (e.g., neutralizing antibodies) that bind to one or more ofthe following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMAor VEGF receptor(s), TRAIL/Apo2, and other bioactive and organicchemical agents, etc. Combinations thereof are also included in theinvention.

A “chemotherapeutic agent” refers to a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®),pegylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin),epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such asmitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), pemetrexed(ALIMTA®); tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone,and 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as folinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2′-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®),albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™),and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g.,ELOXATIN®), and carboplatin; vincas, which prevent tubulinpolymerization from forming microtubules, including vinblastine(VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), andvinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone;leucovorin; novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid, including bexarotene(TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS®or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronicacid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate(AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines,for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID®vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g.,ABARELIX®); BAY439006 (sorafenib, NEXAVAR®; Bayer); SU-11248 (sunitinib,SUTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib oretoricoxib), proteosome inhibitor (e.g. PS341); bortezomib (VELCADE®);CCI-779; tipifarnib (R11577); orafenib, ABT510; Bc1-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (seedefinition below); tyrosine kinase inhibitors (see definition below);serine-threonine kinase inhibitors such as rapamycin (sirolimus,RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636,SARASAR); and pharmaceutically acceptable salts, acids or derivatives ofany of the above; as well as combinations of two or more of the abovesuch as CHOP, an abbreviation for a combined therapy ofcyclophosphamide, doxorubicin, vincristine, and prednisolone; andFOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents as defined herein include “anti-hormonal agents”or “endocrine therapeutics” which act to regulate, reduce, block, orinhibit the effects of hormones that can promote the growth of cancer.They may be hormones themselves, including, but not limited to:anti-estrogens with mixed agonist/antagonist profile, including,tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®),idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, andselective estrogen receptor modulators (SERMs) such as SERM3; pureanti-estrogens without agonist properties, such as fulvestrant(FASLODEX®), and EM800 (such agents may block estrogen receptor (ER)dimerization, inhibit DNA binding, increase ER turnover, and/or suppressER levels); aromatase inhibitors, including steroidal aromataseinhibitors such as formestane and exemestane (AROMASIN®), andnonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®),letrozole (FEMARA®) and aminoglutethimide, and other aromataseinhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®),fadrozole, and 4(5)-imidazoles; lutenizing hormone-releasing hormoneagonists, including leuprolide (LUPRON® and ELIGARD®), goserelin,buserelin, and tripterelin; sex steroids, including progestins such asmegestrol acetate and medroxyprogesterone acetate, estrogens such asdiethylstilbestrol and premarin, and androgens/retinoids such asfluoxymesterone, all transretinoic acid and fenretinide; onapristone;anti-progesterones; estrogen receptor down-regulators (ERDs);anti-androgens such as flutamide, nilutamide and bicalutamide; andpharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above.

An “angiogenic factor or agent” refers to a growth factor whichstimulates the development of blood vessels, e.g., promote angiogenesis,endothelial cell growth, stability of blood vessels, and/orvasculogenesis, etc. For example, angiogenic factors, include, but arenot limited to, e.g., VEGF and members of the VEGF family (VEGF-B,VEGF-C and VEGF-D), PlGF, PDGF family, fibroblast growth factor family(FGFs), TIE ligands (Angiopoietins), ephrins, delta-like ligand 4(DLL4), del-1, fibroblast growth factors: acidic (aFGF) and basic(bFGF), follistatin, granulocyte colony-stimulating factor (G-CSF),hepatocyte growth factor (HGF)/scatter factor (SF), interleukin-8(IL-8), leptin, midkine, neuropilins, placental growth factor,platelet-derived endothelial cell growth factor (PD-ECGF),platelet-derived growth factor, especially PDGF-BB or PDGFR-beta,pleiotrophin (PTN), progranulin, proliferin, transforming growthfactor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta),tumor necrosis factor-alpha (TNF-alpha), etc. It would also includefactors that accelerate wound healing, such as growth hormone,insulin-like growth factor-I (IGF-I), VIGF, epidermal growth factor(EGF), CTGF and members of its family, and TGF-alpha and TGF-beta. See,e.g., Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streitand Detmar (2003) Oncogene 22:3172-3179; Ferrara & Alitalo (1999) NatureMedicine 5(12):1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556(e.g., Table 1 listing known angiogenic factors); and, Sato (2003) Int.J. Clin. Oncol. 8:200-206.

An “anti-angiogenic agent” or “angiogenesis inhibitor” refers to a smallmolecular weight substance, a polynucleotide (including, e.g., aninhibitory RNA (RNAi or siRNA)), a polypeptide, an isolated protein, arecombinant protein, an antibody, or conjugates or fusion proteinsthereof, that inhibits angiogenesis, vasculogenesis, or undesirablevascular permeability, either directly or indirectly. It should beunderstood that the anti-angiogenic agent includes those agents thatbind and block the angiogenic activity of the angiogenic factor or itsreceptor. For example, an anti-angiogenic agent is an antibody or otherantagonist to an angiogenic agent as defined above, e.g., fusionproteins that binds to VEGF-A such as ZALTRAP™ (Aflibercept), antibodiesto VEGF-A such as AVASTIN® (bevacizumab) or to the VEGF-A receptor(e.g., KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors such asGLEEVEC® (Imatinib Mesylate), small molecules that block VEGF receptorsignaling (e.g., PTK787/ZK2284, SU6668, SUTENT®/SU11248 (sunitinibmalate), AMG706, or those described in, e.g., international patentapplication WO 2004/113304). Anti-angiogenic agents also include nativeangiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g.,Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit andDetmar (2003) Oncogene 22:3172-3179 (e.g., Table 3 listinganti-angiogenic therapy in malignant melanoma); Ferrara & Alitalo (1999)Nature Medicine 5(12):1359-1364; Tonini et al. (2003) Oncogene22:6549-6556 (e.g., Table 2 listing known anti-angiogenic factors); and,Sato (2003) Int. J. Clin. Oncol. 8:200-206 (e.g., Table 1 listinganti-angiogenic agents used in clinical trials).

“Docetaxel” refers to1,7β,10β-trihydroxy-9-oxo-5β,20-epoxytax-11-ene-2α,4,13α-triyl 4-acetate2-benzoate13-{(2R,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-3-phenylpropanoate},which, in some embodiments, may be sold under the brand name Taxotere®.In some embodiments, docetaxel is effective for treating at least onecancer selected from breast cancer and non small-cell lung cancer.Nonlimiting exemplary cancers that may be treated with docetaxel includebreast cancer, colorectal cancer, lung cancer, ovarian cancer, prostatecancer, liver cancer, renal cancer, gastric cancer, head and neckcancers, and melanoma.

“Paclitaxel” refers to(2α,4α,5β,7β,10β,13α)-4,10-bis(acetyloxy)-13-{[(2R,3S)-3-(benzoylamino)-2-hydroxy-3-phenylpropanoyl]oxy}-1,7-dihydroxy-9-oxo-5,20-epoxytax-11-en-2-ylbenzoate, which, in some embodiments, may be sold under the brand nameTAXOL®. Nonlimiting exemplary cancers that may be treated withpaclitaxel include breast cancer, lung cancer, and Kaposi's sarcoma.

“Carboplatin” refers tocis-diammine(cyclobutane-1,1-dicarboxylate-O,O′)platinum(II), which, insome embodiments, may be sold under the brand name Paraplatin®.Nonlimiting exemplary cancers that may be treated with paclitaxelinclude, ovarian cancer, non-small cell lung cancer, testicular cancer,stomach cancer, and bladder cancer.

“Oxaliplatin” refers to[(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O′)platinum(II), which,in some embodiments, may be sold under the brand name Eloxatin®.Nonlimiting exemplary cancers that may be treated with oxaliplatininclude colorectal cancer, gastric cancer, and ovarian cancer.

“Cisplatin” refers to (SP-4-2)-diamminedichloridoplatinum. Nonlimitingexemplary cancers that may be treated with cisplatin include sarcomas,small cell lung cancer, ovarian cancer, bladder cancer, testicularcancer, lymphomas, and germ cell tumors.

“Vincristine” refers to methyl(1R,9R,10S,11R,12R,19R)-11-(acetyloxy)-12-ethyl-4-[(13S,15S,17S)-17-ethyl-17-hydroxy-13-(methoxycarbonyl)-1,11-diazatetracyclo[13.3.1.0^(4,12).0^(5,10)]nonadeca-4(12),5,7,9-tetraen-13-yl]-8-formyl-10-hydroxy-5-methoxy-8,16-diazapentacyclo[10.6.1.0^(1,9).0^(2,7).0^(16,19)]nonadeca-2,4,6,13-tetraene-10-carboxylate,which, in some embodiments, may be sold under the brand name Vincasar®.Nonlimiting exemplary cancers that may be treated with vincristineinclude Hodgkin's disease, leukemia, non-Hodgkin's lymphoma,neuroblastoma, rhabdomyosarcoma, acute lymphoblastic leukemia, andWilms' tumor.

“Pemetrexed” refers to(S)-2-[4-[2-(4-amino-2-oxo-3,5,7-triazabicyclo[4.3.0]nona-3,8,10-trien-9-yl)ethyl]benzoyl]aminopentanedioicacid, which, in some embodiments, may be sold under the brand nameAlimta®. Nonlimiting exemplary cancers that may be treated withpemetrexed include non small cell lung cancer, mesothelioma, andesophageal cancer.

“Doxorubicin” refers to(8S,10S)-10-(4-amino-5-hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione,which, in some embodiments, may be sold under the brand nameAdriamycin®. Nonlimiting exemplary cancers that may be treated withdoxorubicin include bladder cancer, breast cancer, lung cancer, ovariancancer, stomach cancer, thyroid cancer, soft tissue sarcoma, multiplemyeloma, Hodgkin's disease, leukemia, non-Hodgkin's lymphoma,neuroblastoma, sarcoma, and Wilms' tumor.

“5-FU” and “5-fluorouracil” refer to 5-fluoro-1H-pyrimidine-2,4-dione,which, in some embodiments, may be sold under the brand name Adrucil®.Nonlimiting exemplary cancers that may be treated with 5-FU includecolorectal cancer, pancreatic cancer, breast cancer, esophageal cancer,gastric cancer, head and neck cancer, hepatoma, and ovarian cancer.

“Leucovorin” is also known as folinic acid, and refers to(S)-2-[4-[(2-amino-5-formyl-4-oxo-5,6,7,8-tetrahydro-1H-pteridin-6-yl)methylamino]benzoyl]aminopentanedioicacid. In some embodiments, leucovorin is administered with5-fluorouracil.

The term “VEGF” or “VEGF-A” as used herein refers to the 165-amino acidhuman vascular endothelial cell growth factor and related 121-, 189-,and 206-amino acid human vascular endothelial cell growth factors, asdescribed by Leung et al. (1989) Science 246:1306, and Houck et al.(1991) Mol. Endocrin, 5:1806, together with the naturally occurringallelic and processed forms thereof. The term “VEGF” also refers toVEGFs from non-human species such as mouse, rat or primate. Sometimesthe VEGF from a specific species are indicated by terms such as hVEGFfor human VEGF, mVEGF for murine VEGF, and etc. The term “VEGF” is alsoused to refer to truncated forms of the polypeptide comprising aminoacids 8 to 109 or 1 to 109 of the 165-amino acid human vascularendothelial cell growth factor. Reference to any such forms of VEGF maybe identified in the present application, e.g., by “VEGF (8-109),” “VEGF(1-109),” “VEGF-A109” or “VEGF165.” The amino acid positions for a“truncated” native VEGF are numbered as indicated in the native VEGFsequence. For example, amino acid position 17 (methionine) in truncatednative VEGF is also position 17 (methionine) in native VEGF. Thetruncated native VEGF has binding affinity for the KDR and Flt-1receptors comparable to native VEGF.

A “VEGF antagonist” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering with VEGFactivities including, but not limited to, its binding to one or moreVEGF receptors. VEGF antagonists include, without limitation, anti-VEGFantibodies and antigen-binding fragments thereof, receptor molecules andderivatives which bind specifically to VEGF thereby sequestering itsbinding to one or more receptors, anti-VEGF receptor antibodies, VEGFreceptor antagonists such as small molecule inhibitors of the VEGFRtyrosine kinases, and immunoadhesins that binds to VEGF such as VEGFtrap (e.g., aflibercept). The term “VEGF antagonist,” as used herein,specifically includes molecules, including antibodies, antibodyfragments, other binding polypeptides, peptides, and non-peptide smallmolecules, that bind to VEGF and are capable of neutralizing, blocking,inhibiting, abrogating, reducing or interfering with VEGF activities.Thus, the term “VEGF activities” specifically includes VEGF mediatedbiological activities of VEGF.

The term “VEGF trap” as used herein means a protein, such as a fusionmolecule, that binds to VEGF and is capable of neutralizing, blocking,inhibiting, abrogating, reducing or interfering with VEGF activities. Anexample of a VEGF trap is aflibercept.

The term “anti-VEGF antibody” or “an antibody that binds to VEGF” refersto an antibody that is capable of binding to VEGF with sufficientaffinity and specificity that the antibody is useful as a diagnosticand/or therapeutic agent in targeting VEGF. Anti-VEGF antibodiessuppress the growth of a variety of human tumor cell lines in nude mice(Kim et al., Nature 362:841-844 (1993); Warren et al., J. Clin. Invest.95:1789-1797 (1995); Borgstrom et al., Cancer Res. 56:4032-4039 (1996);Melnyk et al., Cancer Res. 56:921-924 (1996)) and also inhibitintraocular angiogenesis in models of ischemic retinal disorders. Adamiset al., Arch. Ophthalmol. 114:66-71 (1996). For example, the anti-VEGFantibody can be used as a therapeutic agent in targeting and interferingwith diseases or conditions wherein the VEGF activity is involved. See,e.g., U.S. Pat. Nos. 6,582,959, 6,703,020; WO98/45332; WO 96/30046;WO94/10202, WO2005/044853; EP 0666868B1; US Patent Applications20030206899, 20030190317, 20030203409, 20050112126, 20050186208, and20050112126; Popkov et al., Journal of Immunological Methods 288:149-164(2004); and WO2005012359. The antibody selected will normally have asufficiently strong binding affinity for VEGF. For example, the antibodymay bind hVEGF with a K_(d) value of between 100 nM-1 pM. Antibodyaffinities may be determined by a surface plasmon resonance based assay(such as the BIAcore assay as described in PCT Application PublicationNo. WO2005/012359); enzyme-linked immunoabsorbent assay (ELISA); andcompetition assays (e.g. RIA's), for example. The antibody may besubjected to other biological activity assays, e.g., in order toevaluate its effectiveness as a therapeutic. Such assays are known inthe art and depend on the target antigen and intended use for theantibody. Examples include the HUVEC inhibition assay; tumor cell growthinhibition assays (as described in WO 89/06692, for example);antibody-dependent cellular cytotoxicity (ADCC) and complement-mediatedcytotoxicity (CDC) assays (U.S. Pat. No. 5,500,362); and agonisticactivity or hematopoiesis assays (see WO 95/27062). An anti-VEGFantibody will usually not bind to other VEGF homologues such as VEGF-B,VEGF-C, VEGF-D or VEGF-E, nor other growth factors such as PlGF, PDGF orbFGF.

In one embodiment, anti-VEGF antibodies include a monoclonal antibodythat binds to the same epitope as the monoclonal anti-VEGF antibodyA4.6.1 produced by hybridoma ATCC HB 10709; a recombinant humanizedanti-VEGF monoclonal antibody (see Presta et al. (1997) Cancer Res.57:4593-4599), including but not limited to the antibody known as“bevacizumab” also known as “rhuMAb VEGF” or “AVASTIN®.” AVASTIN® ispresently commercially available. Nonlimiting exemplary cancers that maybe treated with bevacizumab include non-small cell lung cancer,colorectal cancer, breast cancer, renal cancer, ovarian cancer,glioblastoma multiforme, pediatric osteosarcoma, gastric cancer andpancreatic cancer. Bevacizumab comprises mutated human IgG₁ frameworkregions and antigen-binding complementarity-determining regions from themurine antibody A.4.6.1 that blocks binding of human VEGF to itsreceptors. Bevacizumab and other humanized anti-VEGF antibodies arefurther described in U.S. Pat. Nos. 6,884,879, and 7,169,901. Additionalanti-VEGF antibodies are described in PCT Application Publication Nos.WO2005/012359 and WO2009/073160; U.S. Pat. Nos. 7,060,269, 6,582,959,6,703,020; 6,054,297; WO98/45332; WO 96/30046; WO94/10202; EP 0666868B1;U.S. Patent Application Publication Nos. 2006009360, 20050186208,20030206899, 20030190317, 20030203409, and 20050112126; and Popkov etal., Journal of Immunological Methods 288:149-164 (2004).

The terms “subject” and “patient” are used interchangeably herein torefer to a mammal. In some embodiments, the subject or patient is ahuman. In other embodiments, methods of treating other mammals,including, but not limited to, rodents, simians, felines, canines,equines, bovines, porcines, ovines, caprines, mammalian laboratoryanimals, mammalian farm animals, mammalian sport animals, and mammalianpets, are also provided.

“Cancer” and “tumor,” as used herein, are interchangeable terms thatrefer to any abnormal cell or tissue growth or proliferation in ananimal. As used herein, the terms “cancer” and “tumor” encompass solidand hematological/lymphatic cancers and also encompass malignant,pre-malignant, and benign growth, such as dysplasia. Examples of cancerinclude but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia. More particular non-limiting examples of such cancersinclude squamous cell cancer, small-cell lung cancer, pituitary cancer,esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lungcancer, adenocarcinoma of the lung, squamous carcinoma of the lung,cancer of the peritoneum, hepatocellular cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial or uterine carcinoma, salivarygland carcinoma, kidney cancer, renal cancer, liver cancer, prostatecancer, vulval cancer, thyroid cancer, hepatic carcinoma, brain cancer,endometrial cancer, testis cancer, cholangiocarcinoma, gallbladdercarcinoma, gastric cancer, melanoma, and various types of head and neckcancer.

“Treatment,” as used herein, includes any administration or applicationof a therapeutic for condition in a mammal, including a human, andincludes inhibiting the condition or progression of the condition,inhibiting or slowing the condition or its progression, arresting itsdevelopment, partially or fully relieving the condition, or curing thecondition, for example, by causing regression, or restoring or repairinga lost, missing, or defective function; or stimulating an inefficientprocess. In some embodiments, “treatment” refers to clinicalintervention in an attempt to alter the natural course of the individualor cell being treated, and can be performed either for prophylaxis orduring the course of clinical pathology. Desirable effects of treatmentinclude preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis.

An “effective amount” or “therapeutically effective amount” of amolecule or a combination of molecules means an amount that issufficient to treat a condition and/or to inhibit growth of tumor cellsin at least a subset of subjects when given alone or in combination withother treatments. In certain embodiments, a therapeutically effectiveamount refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result. Atherapeutically effective amount of FGFR1 fusion protein of theinvention may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of FGFR1 fusionprotein to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the FGFR1 fusion proteins are outweighed by thetherapeutically beneficial effects. In the case of cancer, the effectiveamount of the drug may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and typically stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and typically stop) tumor metastasis; inhibit, to someextent, tumor growth; allow for treatment of the tumor, and/or relieveto some extent one or more of the symptoms associated with the disorder.To the extent the drug may prevent growth and/or kill existing cancercells, it may be cytostatic and/or cytotoxic.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

The terms “inhibition” or “inhibit” refer to a decrease or cessation ofany phenotypic characteristic or to the decrease or cessation in theincidence, degree, or likelihood of that characteristic. Nonlimitingexemplary inhibition includes inhibition of tumor growth.

Herein, “concurrent” dosing refers to the administration of twotherapeutic molecules within an eight hour time period. In someembodiments, two therapeutic molecules are administered at the sametime. Two therapeutic molecules are considered to be administered at thesame time (i.e. simultaneously) if at least a portion of a dose of eachtherapeutic molecule is administered within 1 hour. Two therapeuticmolecules are administered concurrently if at least one dose isadministered concurrently, even if one or more other doses are notadministered concurrently. In some embodiments, concurrentadministration includes a dosing regimen when the administration of oneor more therapeutic molecule(s) continues after discontinuing theadministration of one or more other therapeutic molecules(s).

Administration “in combination with” one or more further therapeuticagents includes concurrent (including simultaneous) and consecutive(i.e., sequential) administration in any order.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid, or liquid filler, diluent, encapsulating material,formulation auxiliary, or carrier conventional in the art for use with atherapeutic agent that together comprise a “pharmaceutical composition”for administration to a subject. A pharmaceutically acceptable carrieris non-toxic to recipients at the dosages and concentrations employedand is compatible with other ingredients of the formulation. Thepharmaceutically acceptable carrier is appropriate for the formulationemployed. For example, if the therapeutic agent is to be administeredorally, the carrier may be a gel capsule. If the therapeutic agent is tobe administered subcutaneously, the carrier ideally is not irritable tothe skin and does not cause injection site reaction.

Therapeutic Compositions and Methods

Methods of Treating Cancer Using FGFR1 ECDs and/or FGFR1 ECD FusionMolecules in Combination with Other Therapeutic Agents

The invention features the combination of a fibroblast growth factorreceptor 1 (FGFR1) extracellular domain (ECD) or FGFR1 ECD fusionmolecule with one or more additional anti-cancer therapies and the useof such combinations in cancer treatment. Examples of the additionalanti-cancer therapies include, without limitation, surgery, radiationtherapy (radiotherapy), biotherapy, immunotherapy, and chemotherapy or acombination of these therapies. In addition, cytotoxic agents,anti-angiogenic and anti-proliferative agents can be used in combinationwith the FGFR1 ECD or FGFR1 ECD fusion molecule. In certain aspects ofany of the methods and uses, the invention provides treating cancer, byadministering therapeutically effective amounts of an FGFR1 ECD and/orFGFR1 ECD fusion molecule and one or more chemotherapeutic agents to asubject diagnosed with or suffering from a previously untreated cancer.A variety of chemotherapeutic agents may be used in the combinedtreatment methods and uses of the invention. An exemplary andnon-limiting list of chemotherapeutic agents contemplated is providedherein under “Definitions” and in the “Summary of the Invention.” Inanother aspect, the invention provides treating cancer, by administeringtherapeutically effective amounts of an FGFR1 ECD and/or FGFR1 ECDfusion molecule and one or more anti-angiogenic agent(s) to a subjectdiagnosed with a previously untreated cancer. In another aspect, theinvention provides treating cancer, by administering therapeuticallyeffective amounts of an FGFR1 ECD and/or FGFR1 ECD fusion molecule andone or more VEGF antagonists to a subject diagnosed with a previouslyuntreated cancer. In yet another aspect, the invention provides treatingcancer, by administering therapeutically effective amounts of an FGFR1ECD and/or FGFR1 ECD fusion molecule and one or more VEGF antagonists incombination with one or more chemotherapeutic agents to a subjectdiagnosed with a previously untreated cancer. In some embodiments, theone or more VEGF antagonists are anti-VEGF antibodies and/or VEGF traps.

In one example, methods of treating cancer comprising administering to asubject an FGFR1 ECD and/or FGFR1 ECD fusion molecule in combinationwith at least one additional therapeutic agent selected from docetaxel,paclitaxel, vincristine, carboplatin, cisplatin, oxaliplatin,doxorubicin, 5-fluorouracil (5-FU), leucovorin, pemetrexed, sorafenib,etoposide, topotecan, a VEGF antagonist, an anti-VEGF antibody, a VEGFtrap, and bevacizumab are provided. In some embodiments, methods oftreating cancer comprising administering to a subject an FGFR1 ECDand/or FGFR1 ECD fusion molecule and docetaxel are provided. In someembodiments, methods of treating cancer comprising administering to asubject an FGFR1 ECD and/or FGFR1 ECD fusion molecule and pemetrexed areprovided. In some embodiments, methods of treating cancer comprisingadministering to a subject an FGFR1 ECD and/or FGFR1 ECD fusion moleculeand paclitaxel are provided. In some embodiments, methods of treatingcancer comprising administering to a subject an FGFR1 ECD and/or FGFR1ECD fusion molecule and cisplatin are provided. In some embodiments,methods of treating cancer comprising administering to a subject anFGFR1 ECD and/or FGFR1 ECD fusion molecule and vincristine are provided.In some embodiments, methods of treating cancer comprising administeringto a subject an FGFR1 ECD and/or FGFR1 ECD fusion molecule and 5-FU areprovided. In some embodiments, methods of treating cancer comprisingadministering to a subject an FGFR1 ECD and/or FGFR1 ECD fusion moleculeand etoposide are provided. In some embodiments, methods of treatingcancer comprising administering to a subject an FGFR1 ECD and/or FGFR1ECD fusion molecule and topotecan are provided. In some embodiments,methods of treating cancer comprising administering to a subject anFGFR1 ECD and/or FGFR1 ECD fusion molecule and a VEGF antagonist areprovided. In some embodiments, methods of treating cancer comprisingadministering to a subject an FGFR1 ECD and/or FGFR1 ECD fusion moleculeand an anti-VEGF antibody are provided. In some embodiments, methods oftreating cancer comprising administering to a subject an FGFR1 ECDand/or FGFR1 ECD fusion molecule and a VEGF trap are provided. In someembodiments, methods of treating cancer comprising administering to asubject an FGFR1 ECD and/or FGFR1 ECD fusion molecule and bevacizumabare provided. In some embodiments, at least one dose of the FGFR1 ECDand/or FGFR1 ECD fusion molecule and at least one dose of at least oneadditional therapeutic agent are administered concurrently. In someembodiments, at least one dose of the FGFR1 ECD and/or FGFR1 ECD fusionmolecule and at least one dose of at least one additional therapeuticagent are administered at the same time (i.e., simultaneously). In someembodiments, at least one dose of the FGFR1 ECD and/or FGFR1 ECD fusionmolecule and at least one dose of at least two additional therapeuticagents are administered concurrently or simultaneously. In someembodiments, at least one dose of the FGFR1 ECD and/or FGFR1 ECD fusionmolecule and at least one dose of at least three additional therapeuticagents are administered concurrently or simultaneously. In anotherexample, methods of treating cancer comprising administering to asubject an FGFR1-ECD.339-Fc in combination with at least one additionaltherapeutic agent selected from docetaxel, paclitaxel, vincristine,carboplatin, cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU),leucovorin, pemetrexed, and etoposide, topotecan, a VEGF antagonist, ananti-VEGF antibody, a VEGF trap, sorafenib, and bevacizumab areprovided. In some embodiments, methods of treating cancer comprisingadministering to a subject an FGFR1-ECD.339-Fc and docetaxel areprovided. In some embodiments, at least one dose of an FGFR1-ECD.339-Fcand at least one dose of at least one additional therapeutic agent areadministered concurrently. In some embodiments, at least one dose of anFGFR1-ECD.339-Fc and at least one dose of at least one additionaltherapeutic agent are administered at the same time.

Pharmaceutical compositions comprising FGFR1 ECD and/or FGFR1 ECD fusionmolecules (e.g., FGFR1-ECD.339-Fc) are administered in a therapeuticallyeffective amount for the specific indication. The therapeuticallyeffective amount is typically dependent on the weight of the subjectbeing treated, his or her physical or health condition, theextensiveness of the condition to be treated, and/or the age of thesubject being treated. In general, an FGFR1 ECD and/or FGFR1 ECD fusionmolecule (e.g., FGFR1-ECD.339-Fc) is to be administered in an amount inthe range of about 50 μg/kg body weight to about 100 mg/kg body weightper dose. Optionally, the FGFR1 ECD and/or FGFR1 ECD fusion molecule(e.g., FGFR1-ECD.339-Fc) can be administered in an amount in the rangeof about 100 μg/kg body weight to about 30 mg/kg body weight per dose.Further optionally, the FGFR1 ECD and/or FGFR1 ECD fusion molecule(e.g., FGFR1-ECD.339-Fc) can be administered in an amount in the rangeof about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose.In certain embodiments, the FGFR1 ECD and/or FGFR1 ECD fusion molecule(e.g., FGFR1-ECD.339-Fc) is administered at a dose of about 8 mg/kg bodyweight to about 20 mg/kg body weight. In some embodiments, the FGFR1 ECDand/or FGFR1 ECD fusion molecule (e.g., FGFR1-ECD.339-Fc) isadministered at a dose of about 8 mg/kg body weight, about 10 mg/kg bodyweight, about 11 mg/kg body weight, about 12 mg/kg body weight, about 13mg/kg body weight, about 14 mg/kg body weight, about 15 mg/kg bodyweight, about 16 mg/kg body weight, about 17 mg/kg body weight, about 18mg/kg body weight, about 19 mg/kg body weight, or about 20 mg/kg bodyweight. The FGFR1 ECD and/or FGFR1 ECD fusion molecules may also beadministered at ranges from one of the above doses to another. In someembodiments, dosages may be administered twice a week, weekly, everyother week, at a frequency between weekly and every other week, everythree weeks, every four weeks, or every month.

In certain embodiments, dosages of the FGFR1 ECD and/or FGFR1 ECD fusionmolecules can be calculated in two ways depending on the extinctioncoefficient (EC) used. The extinction coefficient differs depending onwhether the glycosylation of the protein is taken into account. In oneembodiment, the extinction coefficient based on the amino acidcomposition of FGFR1-ECD.339-Fc, for example, is 1.42 mL/mg*cm. Inanother embodiment, when the carbohydrate portion as well as the aminoacid portion of FGFR1-ECD.339-Fc is accounted for, the extinctioncoefficient is 1.11 mL/mg*cm. Calculation of the FGFR1-ECD.339-Fc doseusing an EC of 1.11 mL/mg*cm increases the calculated dose by 28%, asshown in Table 1. Although the doses calculated using the two extinctioncoefficients are different, the molar concentrations, or the actualamounts of drug administered, are identical. Unless otherwise noted, thedoses disclosed herein are each calculated using the extinctioncoefficient that does not take account of glycosylation. How thesedosages compare to those calculated using the extinction coefficientthat takes account of glycosylation for FGFR1-ECD.339-Fc is shown inTable 1. As can be seen from Table 1, a dosage of about 8 mg/kg (e.g.,7.8 and 8.0) using an EC of 1.42 mL/mg*cm herein corresponds to a dosageof about 10 mg/kg (e.g. 10.0 and 10.2) when calculated using an EC of1.11 mL/mg*cm. A dosage of about 16 mg/kg (e.g. 15.6 and 16.0 mg/kg)using an EC of 1.42 mL/mg*cm herein corresponds to a dosage of about 20mg/kg (e.g. 20.0 and 20.5) when calculated using an EC of 1.11 mL/mg*cm.As noted in the “Definitions” section above, measured numbers providedherein are approximate and encompass values having additionalsignificant digits that are rounded off. For instance, 8 mg/kgencompasses values with two significant digits such as 7.6, 7.8, 8.0,8.2, 8.4, and 8.45, each of which round to 8. Likewise, a value such as16 mg/kg encompasses values with three significant digits that round to16, such as, for example 15.6 and 16.0.

TABLE 1 Conversion of FGFR1-ECD.339-FC Dose Dose^(a) Dose^(a) EC = 1.42mL/mg*cm EC = 1.11 mL/mg*cm 0.5 0.6 0.75 1.0 1.0 1.3 2.0 2.6 3.0 3.8 4.05.1 5.0 6.4 6.0 7.7 7.0 9.0 7.8 10.0 8.0 10.2 9.0 11.5 10.0 12.8 11.014.1 12.0 15.4 13.0 16.6 14.0 17.9 15.0 19.2 15.6 20.0 16.0 20.5 17.021.8 18.0 23.0 19.0 24.3 20.0 25.6 30.0 38.4 ^(a)Doses shown in mg/kg.

The pharmaceutical compositions comprising FGFR1 ECDs, FGFR1 ECD fusionmolecules, and/or at least one additional therapeutic agent can beadministered as needed to subjects. In certain embodiments, an effectivedose of a therapeutic molecule is administered to a subject one or moretimes. In various embodiments, an effective dose of a therapeuticmolecule is administered to the subject at least once every two months,at least once a month, at least twice a month, once a week, twice aweek, or three times a week. In various embodiments, an effective doseof a therapeutic molecule is administered to the subject for at least aweek, at least a month, at least three months, at least six months, orat least a year.

In certain embodiments, the combined administration of an FGFR1 ECDs,FGFR1 ECD fusion molecule and at least one additional therapeutic agentincludes concurrent administration, including simultaneousadministration, using separate formulations or a single pharmaceuticalformulation, as well as consecutive administration in any order.Optionally there is a time period while both (or all) active agentssimultaneously exert their biological activities. Therapeuticallyeffective amounts of therapeutic agents administered in combination withthe FGFR1 ECD and/or FGFR1 ECD fusion molecule (e.g., FGFR1-ECD.339-Fc)will be at the physician's or veterinarian's discretion. Dosageadministration and adjustment is done to achieve maximal management ofthe conditions to be treated. The dose will additionally depend on suchfactors as the type of therapeutic agent to be used, the specificpatient being treated, the stage of the disease, and the desiredaggressiveness of the treatment regime.

In certain embodiments, a patient is treated with a combination of theFGFR1 ECD and/or FGFR1 ECD fusion molecule (e.g., FGFR1-ECD.339-Fc) anda VEGF antagonist. In some embodiments, the VEGF antagonist is a VEGFtrap (e.g., aflibercept). In some embodiments, the VEGF antagonist is ananti-VEGF antibody. In some embodiments, the VEGF antibody isbevacizumab. One exemplary dosage of bevacizumab is in the range fromabout 0.05 mg/kg to about 20 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0 mg/kg, 4.0 mg/kg, 7.5 mg/kg, 10 mg/kg or 15 mg/kg (or anycombination thereof) may be administered to the patient. Such doses maybe administered intermittently, e.g., every week, every two, or everythree weeks.

In some embodiments, the FGFR1 ECD and/or FGFR1 ECD fusion molecule(e.g., FGFR1-ECD.339-Fc) is administered in combination with anothertherapeutic agent, such as chemotherapeutic agent or anti-angiogenicagent, at the recommended or prescribed dosage and/or frequency of thetherapeutic agent.

Routes of Administration and Carriers

In some embodiments, an FGFR1 ECD and/or FGFR1 ECD fusion molecule canbe administered intravenously and/or subcutaneously. In someembodiments, an FGFR1 ECD and/or FGFR1 ECD fusion molecule can beadministered by another route, such as intra-arterial, parenteral,intranasal, intramuscular, intracardiac, intraventricular,intratracheal, buccal, rectal, intraperitoneal, intradermal, topical,transdermal, or intrathecal, or otherwise by implantation or inhalation.In various embodiments, at least one additional therapeutic agent can beadministered in vivo by a variety of routes, including intravenous,intra-arterial, subcutaneous, parenteral, intranasal, intramuscular,intracardiac, intraventricular, intratracheal, buccal, rectal,intraperitoneal, intradermal, topical, transdermal, and intrathecal, orotherwise by implantation or inhalation. Each of the subjectcompositions can be formulated alone or in combination into preparationsin solid, semi-solid, liquid, or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suppositories,enemas, injections, inhalants, and aerosols.

In various embodiments, compositions comprising an FGFR1 ECD, FGFR1 ECDfusion molecule, and/or at least one additional therapeutic agent areprovided in formulation with pharmaceutically acceptable carriers, awide variety of which are known in the art (see, e.g., Gennaro,Remington: The Science and Practice of Pharmacy with Facts andComparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al.,Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^(th) ed.,Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook ofPharmaceutical Excipients, 3^(rd) ed., Pharmaceutical Press (2000)).Various pharmaceutically acceptable carriers, which include vehicles,adjuvants, carriers, and diluents, are available to the public.Moreover, various pharmaceutically acceptable auxiliary substances, suchas pH adjusting and buffering agents, tonicity adjusting agents,stabilizers, wetting agents and the like, are also available to thepublic. Certain non-limiting exemplary carriers include saline, bufferedsaline, dextrose, water, glycerol, ethanol, and combinations thereof. Insome embodiments, a therapeutic agent is formulated as the brand-namedrug indicated above in the Definitions section, or a genericequivalent. In some embodiments, docetaxel is formulated as Taxotere®(Sanofi Aventis) or a generic equivalent.

In various embodiments, compositions comprising FGFR1 ECDs, FGFR1 ECDfusion molecules, and/or at least one additional therapeutic agent canbe formulated for injection by dissolving, suspending, or emulsifyingthem in an aqueous or nonaqueous solvent, such as vegetable or otheroils, synthetic aliphatic acid glycerides, esters of higher aliphaticacids, or propylene glycol; and if desired, with conventional additivessuch as solubilizers, isotonic agents, suspending agents, emulsifyingagents, stabilizers and preservatives. In various embodiments, thecompositions may be formulated for inhalation, for example, usingpressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen, and the like. The compositions may also beformulated, in various embodiments, into sustained releasemicrocapsules, such as with biodegradable or non-biodegradable polymers.A non-limiting exemplary biodegradable formulation includes poly lacticacid-glycolic acid polymer. A non-limiting exemplary non-biodegradableformulation includes a polyglycerin fatty acid ester. Certain methods ofmaking such formulations are described, for example, in EP 1 125 584 A1.

Pharmaceutical dosage packs comprising one or more containers, eachcontaining one or more doses of an FGFR1 ECD, an FGFR1 ECD fusionmolecule, and/or at least one additional therapeutic agent are alsoprovided. In some embodiments, the dosage packs contain an FGFR1 ECDand/or FGFR1 ECD fusion molecule but do not contain any additionaltherapeutic agent such as docetaxel, paclitaxel, vincristine,carboplatin, cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU),leucovorin, pemetrexed, etoposide, topotecan, sorafenib, a VEGFantagonist, an anti-VEGF antibody, a VEGF trap, or bevacizumab. In otherembodiments, the dosage packs contain at least one additionaltherapeutic agent but do not contain the FGFR1 ECD or FGFR1 ECD fusionmolecule. In other embodiments, the dosage packs contain an FGFR1 ECDand/or FGFR1 ECD fusion molecule and at least one additional therapeuticagent, wherein the FGFR1 ECD and/or FGFR1 ECD fusion molecule is in aseparate container from the at least one additional therapeutic agent.In other embodiments, the FGFR1 ECD and/or FGFR1 ECD fusion molecule isin the same container as the at least one additional therapeutic agent.In certain embodiments where two or more additional therapeutic agentsare supplied, the two or more additional therapeutic agents may be inseparate or in the same containers. In certain embodiments, a unitdosage is provided wherein the unit dosage contains a predeterminedamount of a composition comprising an FGFR1 ECD, an FGFR1 ECD fusionmolecule, and/or at least one additional therapeutic agent with orwithout one or more additional agents. In certain embodiments, such aunit dosage is supplied in single-use prefilled syringe for injection.In various embodiments, the composition contained in the unit dosage maycomprise saline, sucrose, or the like; a buffer, such as phosphate, orthe like; and/or be formulated within a stable and effective pH range.Alternatively, in certain embodiments, the composition may be providedas a lyophilized powder that can be reconstituted upon addition of anappropriate liquid, for example, sterile water. In certain embodiments,a composition comprises one or more substances that inhibit proteinaggregation, including, but not limited to, sucrose and arginine. Incertain embodiments, a composition of the invention comprises heparinand/or a proteoglycan. In some embodiments, a dosage pack comprises anFGFR1 ECD and/or an FGFR1 ECD fusion molecule and/or at least oneadditional therapeutic agent selected from docetaxel, paclitaxel,vincristine, carboplatin, cisplatin, oxaliplatin, doxorubicin,5-fluorouracil (5-FU), leucovorin, pemetrexed, etoposide, topotecan,sorafenib, a VEGF antagonist, an anti-VEGF antibody, a VEGF trap, andbevacizumab.

In some embodiments, a dosage pack further comprises instructions foradministering an FGFR1 ECD and/or an FGFR1 ECD fusion molecule with atleast one additional therapeutic agent to a patient. In someembodiments, the instructions indicate that at least one dose of theFGFR1 ECD and/or the FGFR1 ECD fusion molecule should be administeredconcurrently with the at least one additional therapeutic agent. In someembodiments, the instructions indicate that at least one dose of theFGFR1 ECD and/or the FGFR1 ECD fusion molecule should be administered atthe same time as the at least one additional therapeutic agent.

The term “instructions,” as used herein includes, but is not limited to,labels, package inserts, instructions available in electronic form suchas on a computer readable medium (e.g., a diskette, compact disk, orDVD), instructions available remotely such as over the internet, etc. Adosage pack is considered to include the instructions when the dosagepack provides access to the instructions, a link to the instructions(such as a uniform resource locator, or url), or other mechanism forobtaining a copy of the instructions (such as a return reply card, aphysical address from which instructions may be requested, an e-mailaddress from which instructions may be requested, a phone number thatmay be called to obtain instructions, etc.).

FGFR1 ECDs and FGFR1 ECD Fusion Molecules

Nonlimiting exemplary FGFR1 ECDs include full-length FGFR1 ECDs, FGFR1ECD fragments, and FGFR1 ECD variants. FGFR1 ECDs may include or lack asignal peptide. Exemplary FGFR1 ECDs include, but are not limited to,FGFR1 ECDs having amino acid sequences selected from SEQ ID NOs.: 1, 2,3, and 4.

Non-limiting exemplary FGFR1 ECD fragments include human FGFR1 ECDending at amino acid 339 (counting from the first amino acid of themature form, without the signal peptide). In some embodiments, an FGFR1ECD fragment ends at an amino acid between amino acid 339 and amino acid360 (counting from the first amino acid of the mature form, without thesignal peptide). Exemplary FGFR1 ECD fragments include, but are notlimited to, FGFR1 ECD fragments having amino acid sequences selectedfrom SEQ ID NOs.: 3 and 4.

In some embodiments, an FGFR1 ECD comprises a sequence selected from SEQID NOs: 1 to 4. In some embodiments, an FGFR1 ECD consists of a sequenceselected from SEQ ID NOs: 1 to 4. When an FGFR1 ECD “consists of” asequence selected from SEQ ID NOs: 1 to 4, the FGFR1 ECD may or may notcontain various post-translational modifications, such as glycosylationand sialylation. In other words, when an FGFR1 ECD consists of aparticular amino acid sequence, it does not contain additional aminoacids in the contiguous amino acid sequence, but may containmodifications to amino acid side chains, the N-terminal amino group,and/or the C-terminal carboxy group.

In some embodiments, an FGFR1 ECD fusion molecule comprises a signalpeptide. In some embodiments, an FGFR1 ECD fusion molecule lacks asignal peptide. In some embodiments, the FGFR1 ECD portion of an FGFR1ECD fusion molecule comprises a sequence selected from SEQ ID NOs: 1 to4. In some embodiments, the FGFR1 ECD portion of an FGFR1 ECD fusionmolecule consists of a sequence selected from SEQ ID NOs: 1 to 4. Whenan FGFR1 ECD portion of an FGFR1 ECD fusion molecule “consists of” asequence selected from SEQ ID NOs: 1 to 4, the FGFR1 ECD portion of anFGFR1 ECD fusion molecule may or may not contain variouspost-translational modifications, such as glycosylation and sialylation.In other words, when an FGFR1 ECD portion of an FGFR1 ECD fusionmolecule consists of a particular amino acid sequence, it does notcontain additional amino acids from FGFR1 in the contiguous amino acidsequence, but may contain modifications to amino acid side chains, theN-terminal amino group, and/or the C-terminal carboxy group. Further,because the FGFR1 ECD is linked to a fusion molecule, there may beadditional amino acids a the N- and/or C-terminus of the FGFR1 ECD, butthose amino acids are not from the FGFR1 sequence, but may be from, forexample, a linker sequence, or a fusion partner sequence.

In some embodiments, the fusion partner portion of an FGFR1 ECD fusionmolecule is selected from Fc, albumin, and polyethylene glycol.Nonlimiting exemplary fusion partners are discussed herein.

The inventors have found that administration of an FGFR1 ECD and/or anFGFR1 ECD fusion molecule and at least one additional therapeutic agentselected from docetaxel, paclitaxel, vincristine, carboplatin,cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU), leucovorin,pemetrexed, sorafenib, etoposide, topotecan, a vascular epithelialgrowth factor (VEGF) agonist, a VEGF trap, an anti-VEGF antibody, andbevacizumab is useful for treating cancer. In some embodiments, an FGFR1ECD and/or an FGFR1 ECD fusion molecule is administered with docetaxel.

Fusion Partners and Conjugates

As discussed herein, an FGFR1 ECD may be combined with at least onefusion partner, resulting in an FGFR1 ECD fusion molecule. These fusionpartners may facilitate purification, and the FGFR1 ECD fusion moleculesmay show an increased half-life in vivo. Suitable fusion partners of anFGFR1 ECD include, for example, polymers, such as water solublepolymers, the constant domain of immunoglobulins; all or part of humanserum albumin (HSA); fetuin A; fetuin B; a leucine zipper domain; atetranectin trimerization domain; mannose binding protein (also known asmannose binding lectin), for example, mannose binding protein 1; and anFc region, as described herein and further described in U.S. Pat. No.6,686,179. Nonlimiting exemplary FGFR1 ECD fusion molecules aredescribed, e.g., in U.S. Pat. No. 7,678,890.

An FGFR1 ECD fusion molecule may be prepared by attaching polyaminoacidsor branch point amino acids to the FGFR1 ECD. For example, thepolyaminoacid may be a carrier protein that serves to increase thecirculation half life of the FGFR1 ECD (in addition to the advantagesachieved via a fusion molecule). For the therapeutic purpose of thepresent invention, such polyaminoacids should ideally be those that haveor do not create neutralizing antigenic responses, or other adverseresponses. Such polyaminoacids may be chosen from serum album (such asHSA), an additional antibody or portion thereof, for example the Fcregion, fetuin A, fetuin B, leucine zipper nuclear factor erythroidderivative-2 (NFE2), neuroretinal leucine zipper, tetranectin, or otherpolyaminoacids, for example, lysines. As described herein, the locationof attachment of the polyaminoacid may be at the N terminus or Cterminus, or other places in between, and also may be connected by achemical linker moiety to the selected molecule.

Polymers

Polymers, for example, water soluble polymers, may be useful as fusionpartners to reduce precipitation of the FGFR1 ECD fusion molecule in anaqueous environment, such as typically found in a physiologicalenvironment. Polymers employed in the invention will be pharmaceuticallyacceptable for the preparation of a therapeutic product or composition.

Suitable, clinically acceptable, water soluble polymers include, but arenot limited to, polyethylene glycol (PEG), polyethylene glycolpropionaldehyde, copolymers of ethylene glycol/propylene glycol,monomethoxy-polyethylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, poly (β-aminoacids) (either homopolymers or random copolymers), poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers(PPG) and other polyakylene oxides, polypropylene oxide/ethylene oxidecopolymers, polyoxyethylated polyols (POG) (e.g., glycerol) and otherpolyoxyethylated polyols, polyoxyethylated sorbitol, or polyoxyethylatedglucose, colonic acids or other carbohydrate polymers, Ficoll, ordextran and mixtures thereof.

As used herein, polyethylene glycol (PEG) is meant to encompass any ofthe forms that have been used to derivatize other proteins, such asmono-(C₁-C₁₀) alkoxy- or aryloxy-polyethylene glycol. Polyethyleneglycol propionaldehyde may have advantages in manufacturing due to itsstability in water.

Polymers used herein, for example water soluble polymers, may be of anymolecular weight and may be branched or unbranched. In some embodiments,the polymers have an average molecular weight of between about 2 kDa toabout 100 kDa (the term “about” indicating that in preparations of apolymer, some molecules will weigh more, some less, than the statedmolecular weight). The average molecular weight of each polymer may bebetween about 5 kDa and about 50 kDa, or between about 12 kDa and about25 kDa. Generally, the higher the molecular weight or the more branches,the higher the polymer:protein ratio. Other sizes may also be used,depending on the desired therapeutic profile; for example, the durationof sustained release; the effects, if any, on biological activity; theease in handling; the degree or lack of antigenicity; and other knowneffects of a polymer on an FGFR1 ECD.

Polymers employed in the present invention are typically attached to anFGFR1 ECD with consideration of effects on functional or antigenicdomains of the polypeptide. In general, chemical derivatization may beperformed under any suitable condition used to react a protein with anactivated polymer molecule. Activating groups which can be used to linkthe polymer to the active moieties include sulfone, maleimide,sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane, and5-pyridyl.

Polymers of the invention are typically attached to a heterologouspolypeptide at the alpha (α) or epsilon (ε) amino groups of amino acidsor a reactive thiol group, but it is also contemplated that a polymergroup could be attached to any reactive group of the protein that issufficiently reactive to become attached to a polymer group undersuitable reaction conditions. Thus, a polymer may be covalently bound toan FGFR1 ECD via a reactive group, such as a free amino or carboxylgroup. The amino acid residues having a free amino group may includelysine residues and the N-terminal amino acid residue. Those having afree carboxyl group may include aspartic acid residues, glutamic acidresidues, and the C-terminal amino acid residue. Those having a reactivethiol group include cysteine residues.

Methods for preparing fusion molecules conjugated with polymers, such aswater soluble polymers, will each generally involve (a) reacting anFGFR1 ECD with a polymer under conditions whereby the polypeptidebecomes attached to one or more polymers and (b) obtaining the reactionproduct. Reaction conditions for each conjugation may be selected fromany of those known in the art or those subsequently developed, butshould be selected to avoid or limit exposure to reaction conditionssuch as temperatures, solvents, and pH levels that would inactivate theprotein to be modified. In general, the optimal reaction conditions forthe reactions will be determined case-by-case based on known parametersand the desired result. For example, the larger the ratio ofpolymer:polypeptide conjugate, the greater the percentage of conjugatedproduct. The optimum ratio (in terms of efficiency of reaction in thatthere is no excess unreacted polypeptide or polymer) may be determinedby factors such as the desired degree of derivatization (e.g., mono-,di-, tri-, etc.), the molecular weight of the polymer selected, whetherthe polymer is branched or unbranched and the reaction conditions used.The ratio of polymer (for example, PEG) to a polypeptide will generallyrange from 1:1 to 100:1. One or more purified conjugates may be preparedfrom each mixture by standard purification techniques, including amongothers, dialysis, salting-out, ultrafiltration, ion-exchangechromatography, gel filtration chromatography, and electrophoresis.

One may specifically desire an N-terminal chemically modified FGFR1 ECD.One may select a polymer by molecular weight, branching, etc., theproportion of polymers to FGFR1 ECD molecules in the reaction mix, thetype of reaction to be performed, and the method of obtaining theselected N-terminal chemically modified FGFR1 ECD. The method ofobtaining the N-terminal chemically modified FGFR1 ECD preparation(separating this moiety from other monoderivatized moieties ifnecessary) may be by purification of the N-terminal chemically modifiedFGFR1 ECD material from a population of chemically modified proteinmolecules.

Selective N-terminal chemical modification may be accomplished byreductive alkylation which exploits differential reactivity of differenttypes of primary amino groups (lysine versus the N-terminal) availablefor derivatization in a particular protein. Under the appropriatereaction conditions, substantially selective derivatization of theprotein at the N terminus with a carbonyl group-containing polymer isachieved. For example, one may selectively attach a polymer to the Nterminus of the protein by performing the reaction at a pH that allowsone to take advantage of the pKa differences between the ε-amino groupof the lysine residues and that of the α-amino group of the N-terminalresidue of the protein. By such selective derivatization, attachment ofa polymer to a protein is controlled: the conjugation with the polymertakes place predominantly at the N terminus of the protein and nosignificant modification of other reactive groups, such as the lysineside chain amino groups, occurs. Using reductive alkylation, the polymermay be of the type described above and should have a single reactivealdehyde for coupling to the protein. Polyethylene glycolpropionaldehyde, containing a single reactive aldehyde, may also beused.

In one embodiment, the present invention contemplates the chemicallyderivatized FGFR1 ECD to include mono- or poly- (e.g., 2-4) PEGmoieties. Pegylation may be carried out by any of the pegylationreactions available. Methods for preparing a pegylated protein productwill generally include (a) reacting a polypeptide with polyethyleneglycol (such as a reactive ester or aldehyde derivative of PEG) underconditions whereby the protein becomes attached to one or more PEGgroups; and (b) obtaining the reaction product(s). In general, theoptimal reaction conditions will be determined case by case based onknown parameters and the desired result.

There are a number of PEG attachment methods known in the art. See, forexample, EP 0 401 384; Malik et al., Exp. Hematol., 20:1028-1035 (1992);Francis, Focus on Growth Factors, 3(2):4-10 (1992); EP 0 154 316; EP 0401 384; WO 92/16221; WO 95/34326; and the other publications citedherein that relate to pegylation.

Pegylation may be carried out, e.g., via an acylation reaction or analkylation reaction with a reactive polyethylene glycol molecule. Thus,protein products according to the present invention include pegylatedproteins wherein the PEG group(s) is (are) attached via acyl or alkylgroups. Such products may be mono-pegylated or poly-pegylated (forexample, those containing 2-6 or 2-5 PEG groups). The PEG groups aregenerally attached to the protein at the α- or ε-amino groups of aminoacids, but it is also contemplated that the PEG groups could be attachedto any amino group attached to the protein that is sufficiently reactiveto become attached to a PEG group under suitable reaction conditions.

Pegylation by acylation generally involves reacting an active esterderivative of polyethylene glycol (PEG) with an FGFR1 ECD. For acylationreactions, the polymer(s) selected typically have a single reactiveester group. Any known or subsequently discovered reactive PEG moleculemay be used to carry out the pegylation reaction. An example of asuitable activated PEG ester is PEG esterified to N-hydroxysuccinimide(NHS). As used herein, acylation is contemplated to include, withoutlimitation, the following types of linkages between the therapeuticprotein and a polymer such as PEG: amide, carbamate, urethane, and thelike, see for example, Chamow, Bioconjugate Chem., 5:133-140 (1994).Reaction conditions may be selected from any of those currently known orthose subsequently developed, but should avoid conditions such astemperature, solvent, and pH that would inactivate the polypeptide to bemodified.

Pegylation by acylation will generally result in a poly-pegylatedprotein. The connecting linkage may be an amide. The resulting productmay be substantially only (e.g., >95%) mono-, di-, or tri-pegylated.However, some species with higher degrees of pegylation may be formed inamounts depending on the specific reaction conditions used. If desired,more purified pegylated species may be separated from the mixture(particularly unreacted species) by standard purification techniques,including among others, dialysis, salting-out, ultrafiltration,ion-exchange chromatography, gel filtration chromatography, andelectrophoresis.

Pegylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with a polypeptide in the presence of a reducingagent. For the reductive alkylation reaction, the polymer(s) selectedshould have a single reactive aldehyde group. An exemplary reactive PEGaldehyde is polyethylene glycol propionaldehyde, which is water stable,or mono C₁-C₁₀ alkoxy or aryloxy derivatives thereof, see for example,U.S. Pat. No. 5,252,714.

Markers

Moreover, FGFR1 ECDs of the present invention may be fused to markersequences, such as a peptide that facilitates purification of the fusedpolypeptide. The marker amino acid sequence may be a hexa-histidinepeptide such as the tag provided in a pQE vector (Qiagen, Mississauga,Ontario, Canada), among others, many of which are commerciallyavailable. As described in Gentz et al., Proc. Natl. Acad. Sci.86:821-824 (1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. Another peptide tag useful forpurification, the hemagglutinin (HA) tag, corresponds to an epitopederived from the influenza HA protein. (Wilson et al., Cell 37:767(1984)). Any of these above fusions may be engineered using the FGFR1ECDs described herein.

Oligomerization Domain Fusion Partners

In various embodiments, oligomerization offers some functionaladvantages to a fusion protein, including, but not limited to,multivalency, increased binding strength, and the combined function ofdifferent domains. Accordingly, in some embodiments, a fusion partnercomprises an oligomerization domain, for example, a dimerization domain.Exemplary oligomerization domains include, but are not limited to,coiled-coil domains, including alpha-helical coiled-coil domains;collagen domains; collagen-like domains; and certain immunoglobulindomains. Exemplary coiled-coil polypeptide fusion partners include, butare not limited to, the tetranectin coiled-coil domain; the coiled-coildomain of cartilage oligomeric matrix protein; angiopoietin coiled-coildomains; and leucine zipper domains. Exemplary collagen or collagen-likeoligomerization domains include, but are not limited to, those found incollagens, mannose binding lectin, lung surfactant proteins A and D,adiponectin, ficolin, conglutinin, macrophage scavenger receptor, andemilin.

Antibody Fc Immunoglobulin Domain Fusion Partners

Many Fc domains that may be used as fusion partners are known in theart. In some embodiments, a fusion partner is an Fc immunoglobulindomain. An Fc fusion partner may be a wild-type Fc found in a naturallyoccurring antibody, a variant thereof, or a fragment thereof.Non-limiting exemplary Fc fusion partners include Fcs comprising a hingeand the CH2 and CH3 constant domains of a human IgG, for example, humanIgG1, IgG2, IgG3, or IgG4. Additional exemplary Fc fusion partnersinclude, but are not limited to, human IgA and IgM. In some embodiments,an Fc fusion partner comprises a C237S mutation, for example, in an IgG1(see, for example, SEQ ID NO: 8). In some embodiments, an Fc fusionpartner comprises a hinge, CH2, and CH3 domains of human IgG2 with aP331S mutation, as described in U.S. Pat. No. 6,900,292. Certainexemplary Fc domain fusion partners are shown in SEQ ID NOs: 8 to 10.

Albumin Fusion Partners and Albumin-Binding Molecule Fusion Partners

In some embodiments, a fusion partner is an albumin. Exemplary albuminsinclude, but are not limited to, human serum album (HSA) and fragmentsof HSA that are capable of increasing the serum half-life orbioavailability of the polypeptide to which they are fused. In someembodiments, a fusion partner is an albumin-binding molecule, such as,for example, a peptide that binds albumin or a molecule that conjugateswith a lipid or other molecule that binds albumin. In some embodiments,a fusion molecule comprising HSA is prepared as described, e.g., in U.S.Pat. No. 6,686,179.

Exemplary Attachment of Fusion Partners

The fusion partner may be attached, either covalently or non-covalently,to the N terminus or the C terminus of the FGFR1 ECD. The attachment mayalso occur at a location within the FGFR1 ECD other than the N terminusor the C terminus, for example, through an amino acid side chain (suchas, for example, the side chain of cysteine, lysine, serine, orthreonine).

In either covalent or non-covalent attachment embodiments, a linker maybe included between the fusion partner and the FGFR1 ECD. Such linkersmay be comprised of at least one amino acid or chemical moiety.Exemplary methods of covalently attaching a fusion partner to an FGFR1ECD include, but are not limited to, translation of the fusion partnerand the FGFR1 ECD as a single amino acid sequence and chemicalattachment of the fusion partner to the FGFR1 ECD. When the fusionpartner and an FGFR1 ECD are translated as single amino acid sequence,additional amino acids may be included between the fusion partner andthe FGFR1 ECD as a linker. In some embodiments, the linker is selectedbased on the polynucleotide sequence that encodes it, to facilitatecloning the fusion partner and/or FGFR1 ECD into a single expressionconstruct (for example, a polynucleotide containing a particularrestriction site may be placed between the polynucleotide encoding thefusion partner and the polynucleotide encoding the FGFR1 ECD, whereinthe polynucleotide containing the restriction site encodes a short aminoacid linker sequence). When the fusion partner and the FGFR1 ECD arecovalently coupled by chemical means, linkers of various sizes maytypically be included during the coupling reaction.

Exemplary methods of non-covalently attaching a fusion partner to anFGFR1 ECD include, but are not limited to, attachment through a bindingpair. Exemplary binding pairs include, but are not limited to, biotinand avidin or streptavidin, an antibody and its antigen, etc.

Co-Translational and Post-Translational Modifications

The invention encompasses administration of FGFR1 ECDs and FGFR1 ECDfusion molecules that are differentially modified during or aftertranslation, for example by glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, or linkage to an antibody molecule or othercellular ligand. Any of numerous chemical modifications may be carriedout by known techniques, including, but not limited to, specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease; NABH₄, acetylation; formylation; oxidation; reduction; and/ormetabolic synthesis in the presence of tunicamycin.

Additional post-translational modifications encompassed by the inventioninclude, for example, for example, N-linked or O-linked carbohydratechains, processing of N-terminal or C-terminal ends), attachment ofchemical moieties to the amino acid backbone, chemical modifications ofN-linked or O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of prokaryotic host cellexpression. A nonlimiting discussion of various post-translationalmodifications of FGFR1 ECDs and FGFR1 ECD fusion molecules can be found,e.g., in U.S. Pat. No. 7,678,890.

FGFR1 ECD and FGFR1 ECD Fusion Molecule Expression and ProductionVectors

Vectors comprising polynucleotides that encode FGFR1 ECDs are provided.Vectors comprising polynucleotides that encode FGFR1 ECD fusionmolecules are also provided. Such vectors include, but are not limitedto, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc.

In some embodiments, a vector is selected that is optimized forexpression of polypeptides in CHO or CHO-derived cells. Exemplary suchvectors are described, e.g., in Running Deer et al., Biotechnol. Prog.20:880-889 (2004).

In some embodiments, a vector is chosen for in vivo expression of FGFR1ECDs and/or FGFR1 ECD fusion molecules in animals, including humans. Insome such embodiments, expression of the polypeptide is under thecontrol of a promoter that functions in a tissue-specific manner. Forexample, liver-specific promoters are described, e.g., in PCTPublication No. WO 2006/076288. A nonlimiting discussion of variousexpression vectors can be found, e.g., in U.S. Pat. No. 7,678,890.

Host Cells

In various embodiments, FGFR1 ECDs or FGFR1 ECD fusion molecules may beexpressed in prokaryotic cells, such as bacterial cells; or ineukaryotic cells, such as fungal cells, plant cells, insect cells, andmammalian cells. Such expression may be carried out, for example,according to procedures known in the art. Exemplary eukaryotic cellsthat may be used to express polypeptides include, but are not limitedto, COS cells, including COS 7 cells; 293 cells, including 293-6E cells;CHO cells, including CHO-S and DG44 cells; and NSO cells. In someembodiments, a particular eukaryotic host cell is selected based on itsability to make certain desired post-translational modifications to theFGFR1 ECDs or FGFR1 ECD fusion molecules. For example, in someembodiments, CHO cells produce FGFR1 ECDs and/or FGFR1 ECD fusionmolecules that have a higher level of sialylation than the samepolypeptide produced in 293 cells.

Introduction of a nucleic acid into a desired host cell may beaccomplished by any method known in the art, including but not limitedto, calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, etc. Nonlimiting exemplary methods are described, e.g., inSambrook et al., Molecular Cloning, A Laboratory Manual, 3^(rd) ed. ColdSpring Harbor Laboratory Press (2001). Nucleic acids may be transientlyor stably transfected in the desired host cells, according to methodsknown in the art. A nonlimiting discussion of host cells and methods ofpolypeptides in host cells can be found, e.g., in U.S. Pat. No.7,678,890.

In some embodiments, a polypeptide may be produced in vivo in an animalthat has been engineered or transfected with a nucleic acid moleculeencoding the polypeptide, according to methods known in the art.

Purification of FGFR1 ECD Polypeptides

FGFR1 ECDs or FGFR1 ECD fusion molecules may be purified by variousmethods known in the art. Such methods include, but are not limited to,the use of affinity matrices or hydrophobic interaction chromatography.Suitable affinity ligands include any ligands of the FGFR1 ECD or of thefusion partner. Suitable affinity ligands in the case of an antibodythat binds FGFR1 include, but are not limited to, FGFR1 itself andfragments thereof. Further, a Protein A, Protein G, Protein A/G, or anantibody affinity column may be used to bind to an Fc fusion partner topurify an FGFR1 ECD fusion molecule. Antibodies to FGFR1 ECD may also beused to purify FGFR1 ECD or FGFR1 ECD fusion molecules. Hydrophobicinteractive chromatography, for example, a butyl or phenyl column, mayalso suitable for purifying some polypeptides. Many methods of purifyingpolypeptides are known in the art. A nonlimiting discussion of variousmethods of purifying polypeptides can be found, e.g., in U.S. Pat. No.7,678,890.

EXAMPLES

The examples discussed below are intended to be purely exemplary of theinvention and should not be considered to limit the invention in anyway. The examples are not intended to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (for example, amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1: Administration of FGFR1-ECD.339-Fc and Docetaxel in the111703 Non-Small Cell Lung Cancer (NSCLC) Xenograft Model

Six week old female SCID mice were purchased from Charles RiverLaboratories (Wilmington, Mass.) and were acclimated for 1 week beforethe start of the study. Human non-small cell lung cancer (NSCLC) cellline H1703 was used as the tumor model and was purchased from ATCC(Manassas, Va.; Cat. No. CRL-5889). The cells were cultured for threepassages in RPMI+10% FBS+1% L-glutamine at 37° C. in a humidifiedatmosphere with 5% CO₂. When the cultured cells reached 85-90%confluence, cells were harvested and resuspended in cold Ca²⁺ and Mg²⁺free phosphate buffered saline (PBS) containing 50% Matrigel at 2.5×10⁷cells per milliliter. The cells were implanted subcutaneously over theright flank of the mice at 2.5×10⁶ cells/100 μl/mouse. One day aftertumor implantation, mice were randomized according to body weight at 10mice per group.

FGFR1-ECD.339-Fc was formulated in PBS at 3 mg/ml and administeredintraperitoneally (i.p.) at 15 mg/kg (300 μg/100 μl/mouse) twice a weekfor four weeks. Docetaxel was purchased from Toronto Research Chemicals(North York, Ontario, Canada; cat. No. D494420) and formulated in H₂Ocontaining 5% Tween 80 and 5% glucose. Docetaxel was administered i.p.at 25 mg/kg (equivalent to about 75 mg/m² in a human) once every threeweeks for two doses. In combination groups, FGFR1-ECD.339-Fc anddocetaxel were either given concurrently or sequentially withFGFR1-ECD.339-Fc given one day before docetaxel or vice versa. Humanalbumin was purchased from Grifols USA (Los Angeles, Calif.; Cat. No.NDC 61953-0002-1), formulated in PBS at 3 mg/mL and was used as negativecontrol at 300 μg/100 μL/mouse (15 mg/kg). The dosing schedule for eachset of mice is shown in Table 2.

TABLE 2 Dosing Schedule Group n Route Treatment Schedule 1 10 i.p.Albumin 2×/wk × 6 wks 2 10 i.p. FGFR1-ECD.339-Fc 2×/wk × 6 wks 3 10 i.p.Docetaxel 1×/3 wk × 2 doses 4 10 i.p. FGFR1-ECD.339-Fc 2×/wk × 6 wksi.p. Docetaxel 1×/3 wk × 2 doses 5 10 i.p. FGFR1-ECD.339-Fc 2×/wk × 6wks i.p. Docetaxel (1 day after) 1×/3 wk × 2 doses 6 10 i.p.FGFR1-ECD.339-Fc 2×/wk × 6 wks i.p. Docetaxel (1 day before) 1×/3 wk × 2doses

The tumor volume and body weight of the mice were monitored twice a weekthroughout the study. The tumor volume was measured by external caliperto determine the greatest longitudinal diameter (length) and thegreatest transverse diameter (width). The tumor volume was thencalculated using the following formula:

Tumor volume (mm³)=(length×width²)/2

On day 38, when the average tumor volume in the Albumin group reached850 mm³, the mice were euthanized by isoflurane inhalation and cervicaldislocation.

The mean tumor volume throughout the study for each set of mice is shownin FIG. 1. In that experiment, sequential administration ofFGFR1-ECD.339-Fc and docetaxel inhibited tumor growth more than eitherdrug alone. Furthermore, in that experiment, concurrent administrationof FGFR1-ECD.339-Fc and docetaxel was more effective than sequentialadministration at inhibiting tumor growth. No weight loss was observedover the course of the study. (Data not shown.)

The tumor volume of each group of mice on day 38 was analyzed by one-wayANOVA followed by Tukey's test. The results of that analysis are shownin Table 3.

TABLE 3 Tumor volume analysis on day 38 Mean Tumor Tumor p Value comparevolume growth to control mm³(±SD) inhibition (%) (Tukey's test) Albumin835 (±151) — — FGFR1-ECD.339-Fc 557 (±151) 33 <0.01 Docetaxel 209 (±303)74 <0.001 FGFR1-ECD.339-  27 (±55)  96 <0.001 Fc/Docetaxel concurrentFGFR1-ECD.339-  99 (±117) 88 <0.001 Fc/Docetaxel SequentialDocetaxel/FGFR1-  72 (±109) 91 <0.001 ECD.339-Fc Sequential

Administration of FGFR1-ECD.339-Fc alone resulted in 33% (p<0.01) tumorgrowth inhibition, and administration of docetaxel alone resulted in 74%(p<0.001) tumor growth inhibition. Sequential administration withFGFR1-ECD.339-Fc first resulted in 88% (p<0.001) tumor growthinhibition, and sequential administration with docetaxel first resultedin 91% (p<0.001) tumor growth inhibition. Finally, concurrent dosing ofFGFR1-ECD.339-Fc and docetaxel resulted in 96% (p<0.001) tumor growthinhibition.

Fractional tumor volume analysis was used to assess the degree ofenhanced (additive or synergistic) or decreased (antagonistic) tumorgrowth inhibition in the sequential and concurrent combinations ofFGFR1-ECD.339-Fc and docetaxel. The results of that analysis are shownin Table 4.

TABLE 4 Analysis of fractional tumor volume^(a) on day 38 FGFR1-Expected/ ECD.339-Fc Docetaxel Expected^(b) Observed Observed^(c)Concurrent 0.68 0.25 0.17 0.03 5.67 FGFR1- 0.68 0.25 0.17 0.11 1.55ECD.339-Fc/ Docetaxel sequential Docetaxel/ 0.68 0.25 0.17 0.09 1.89FGFR1- ECD.339-Fc sequential ^(a)Fractional tumor volume (FTV) = (Meantumor volume (TV) treated)/(Mean TV control) ^(b)Expected = (FTV drug 1)× (FTV drug 2) ^(c)Ratio of expected over observed, >2 = synergistic; ~1= additive; <0.5 = antagonistic.

Those results demonstrate that concurrent administration ofFGFR1-ECD.339-Fc and docetaxel results in synergistic inhibition oftumor growth, while sequential administration of FGFR1-ECD.339-Fc anddocetaxel results in additive inhibition.

Example 2: Administration of FGFR1-ECD.339-Fc and Pemetrexed in the11520 Non-Small Cell Lung Cancer (NSCLC) Xenograft Model

Six to eight week old female SCID mice were purchased from Charles RiverLaboratories (Wilmington, Mass.) and were acclimated for 1 week beforethe start of the study. Squamous cell lung cancer cell line NCI-H520 wasused as the tumor model and was purchased from ATCC (Manassas, Va.; Cat.No. HTB-182). The cells were cultured for three to four passages inRPMI+10% FBS+1% L-glutamine at 37° C. in a humidified atmosphere with 5%CO₂. When the cultured cells reached 85-90% confluence, cells wereharvested and resuspended in cold Ca²⁺ and Mg²⁺ free PBS containing 50%Matrigel at 3.5×10⁷ cells/ml. The cells were implanted subcutaneouslyover the right flank of the mice at 3.5×10⁶ cells/100 μl/mouse. One dayafter tumor implantation, mice were randomized according to body weightat 10 mice per group. Dosing for all groups began one day post tumorimplantation.

FGFR1-ECD.339-Fc was formulated in PBS at 3 mg/ml and administeredintraperitoneally (i.p.) at 15 mg/kg (300 μg/100 μl/mouse) twice a weekfor six weeks. Pemetrexed disodium was purchased from Fisher Scientific(Pittsburgh, Pa.; Cat. No. NC9564691) and formulated in Saline USP at12.5 mg/mL, 25 mg/mL, and 50 mg/mL. Pemetrexed was administered i.p. atthree different dosage levels: 62.6 mg/kg (1.25 mg/100 μl/mouse); 125mg/kg (2.5 mg/100 μl/mouse); and 250 mg/kg (5 mg/100 μl/mouse) daily forfive days in week one, and daily for five days in week 2. In combinationgroups, FGFR1-ECD.339-Fc and pemetrexed were administered on the sameschedule as when given as single agents, and were administeredconcurrently on days 1, 4, 8, and 11. Human albumin was purchased fromGrifols USA (Los Angeles, Calif.; Cat. No. NDC 61953-0002-1), formulatedin PBS at 3 mg/mL and was used as negative control at 300 μg/100μL/mouse (15 mg/kg). The dosing schedule for each group of mice is shownin Table 5.

TABLE 5 Dosing Schedule Group n Route Treatment Dose Schedule 1 10 i.p.Albumin 15 mg/kg 2×/wk × 6 wks 2 10 i.p. FGFR1- 15 mg/kg 2×/wk × 6 wksECD.339-Fc 3 10 i.p. Pemetrexed 62.5 mg/kg QD × 5 × 2 cycles 4 10 i.p.Pemetrexed 125 mg/kg QD × 5 × 2 cycles 5 10 i.p. Pemetrexed 250 mg/kg QD× 5 × 2 cycles 6 10 i.p. FGFR1- 15 mg/kg 2×/wk × 6 wks ECD.339-Fc 62.5mg/kg QD × 5 × 2 i.p. Pemetrexed cycles 7 10 i.p. FGFR1- 15 mg/kg 2×/wk× 6 wks ECD.339-Fc 125 mg/kg QD × 5 × 2 i.p. Pemetrexed cycles 8 10 i.p.FGFR1- 15 mg/kg 2×/wk × 6 wks ECD.339-Fc 250 mg/kg QD × 5 × 2 i.p.Pemetrexed cycles

The tumor volume and body weight of the mice were monitored twice a weekthroughout study. The tumor volume was measured and calculated using themethod and formula described in Example 1.

Animals were euthanized when any of the following signs were observedbefore the end of the study: body weight loss of ≧15% of initial bodyweight; tumor ulceration of ≧30% of tumor surface area; mice weremoribund; or the individual tumor volume was ≧2000 mm³. The mice wereeuthanized by isoflurane inhalation and cervical dislocation.

The mean tumor volume throughout the study for low pemetrexed dosagegroups, medium pemetrexed dosage groups, and high pemetrexed dosagegroups are shown in FIGS. 2A, 3A, and 4A, respectively. FIGS. 2B, 3B,and 4B show the body weight of the mice in each group over the course ofthe study. It appears that 250 mg/kg pemetrexed is approaching themaximum tolerated dose in the mice, based on the loss of body weightfollowing administration of that dose. See FIG. 4B.

Administration of either FGFR1-ECD.339-Fc or pemetrexed at the low ormedium dose alone resulted in tumor inhibition over the course of thestudy. The highest dose of pemetrexed alone did not appear to inhibittumor growth. Concurrent dosing of FGFR1-ECD.339-Fc and pemetrexedresulted in greater inhibition of tumor growth, although the tumorinhibition observed with the combination of FGFR1-ECD.339-Fc and thehighest dose of pemetrexed was not statistically significant.

The body weight graphs show that the mice tolerated the low and mediumdoses of pemetrexed well. At the high dose of pemetrexed, the miceinitially lost significant body weight. The dosing was therefore stoppedafter the first 5-day dosing. Thus, in the high dose group, the animalsonly received the first five doses and missed the second five doses.

The tumor volume of each group of mice on day 49 was analyzed by one-wayANOVA followed by Tukey's test. The results of that analysis are shownin Table 6.

TABLE 6 Tumor volume analysis on day 49 Tumor p Value Mean Tumor growthcompare volume inhibition to control mm³ (±SD) (%) (Tukey's test)Albumin 917.3 (±313.9) — — FGFR1-ECD.339-Fc 630.4 (±461.5) 31 >0.05Pemetrexed 62.5 mg/kg 544.7 (±341.5) 40 >0.05 FGFR1-ECD.339-Fc/ 292.5(±198.5) 68 <0.01 Pemetrexed 62.5 mg/kg Pemetrexed 125 mg/kg 689.5(±328.7) 24 >0.05 FGFR1-ECD.339-Fc/ 389.2 (±271.3) 57 <0.05 Pemetrexed125 mg/kg Pemetrexed 250 mg/kg 926.6 (±339.9) 1 >0.05 FGFR1-ECD.339-Fc/489.3 (±231.0) 46 >0.05 Pemetrexed 250 mg/kg

Administration of FGFR1-ECD.339-Fc alone resulted in 31% (p>0.05) tumorgrowth inhibition, and administration of pemetrexed alone at 62.5, 125or 250 mg/kg resulted in 40, 24 or 1% (p>0.05) tumor growth inhibitionrespectively. Concurrent dosing of FGFR1-ECD.339-Fc (15 mg/kg) andpemetrexed at 62.5, 125, or 250 mg/kg resulted in 68% (p<0.01), 57%(p<0.05) or 46% (p>0.05) tumor growth inhibition respectively.

Fractional tumor volume analysis was used to assess the degree ofenhanced (additive or synergistic) or decreased (antagonistic) tumorgrowth inhibition following administration of FGFR1-ECD.339-Fc andpemetrexed. The results of that analysis are shown in Table 7.

TABLE 7 Analysis of fractional tumor volume^(a) on day 49 FGFR1- Ex- Ob-Expected/ ECD.339-Fc Pemetrexed pected^(b) served Observed^(c)Pemetrexed 0.68 0.59 0.40 0.31 1.29 62.5 Pemetrexed 0.68 0.75 0.51 0.421.21 125 Pemetrexed 0.68 1.00 0.68 0.53 1.28 250 ^(a)Fractional tumorvolume (FTV) = (Mean tumor volume (TV) treated)/(Mean TV control)^(b)Expected = (FTV drug 1) × (FTV drug 2) ^(c)Ratio of expected overobserved, >2 = synergistic; ~1 = additive; <0.5 = antagonistic.

Those results demonstrate that administration of FGFR1-ECD.339-Fc andpemetrexed results in additive inhibition of tumor growth.

Example 3: Administration of FGFR1-ECD.339-Fc in Combination withVarious Chemotherapeutics in the A549 Non-Small Cell Lung Cancer (NSCLC)Xenograft Model

Six weeks old female SCID mice were purchased from Charles RiverLaboratories (Wilmington, Mass.) and were acclimated for 1 week beforethe start of the study. A549 cells purchased from ATCC (Manassas, Va.;Cat. No. CCL-185) were cultured for three passages in RPMI+10% FBS+1%L-glutamine at 37° C. in a humidified atmosphere with 5% CO₂. When thecultured cells reached 85-90% confluence, cells were harvested andresuspended in cold Ca′ and Mg′ free phosphate buffered saline (PBS)containing 50% Matrigel at 5×10⁷ cells per milliliter. The cells wereimplanted subcutaneously over the right flank of the mice at 5×10⁶cells/100 μl/mouse. One day after tumor implantation, mice wererandomized according to body weight at 10 mice per group.

The tumor volume and body weight of the mice were monitored twice a weekthroughout each study. The tumor volume was measured and calculatedusing the method and formula described in Example 1.

The tumor volume of each group of mice at the end of each study wasanalyzed by one-way ANOVA followed by Tukey's test. Fractional tumorvolume analysis was then used to assess the degree of enhanced (additiveor synergistic) or decreased (antagonistic) tumor growth inhibitionachieved following administration of FGFR1-ECD.339-Fc with one or moreadditional chemotherapeutic molecules.

Certain study details, and the results for each combination, arediscussed below.

A. FGFR1-ECD.339-Fc and Cisplatin

FGFR1-ECD.339-Fc was formulated in 0.9% Sodium Chloride Injection USP(Henry Schein, Inc., Melville, N.Y.; Cat. No. 1533826) at 4 mg/ml andadministered intraperitoneally (i.p.) at 20 mg/kg (400 μg/100 μl/mouse)twice per week for six weeks. Cisplatin was purchased from Sigma-Aldrich(St. Louis, Mo.; Cat. No. P4394), formulated in 0.9% saline, andadministered i.p. at 3.5 mg/kg (17 μg/100 μl per mouse) once per weekfor six weeks. Saline was used as negative control and was administeredi.p. at 100 μl per mouse twice a week for six weeks.

On day 42, when the average tumor volume in the vehicle group reached1300 mm³, the mice were euthanized by isoflurane inhalation and cervicaldislocation.

The mean tumor volume throughout the study for each set of mice is shownin FIG. 5. In that experiment, administration of FGFR1-ECD.339-Fc andcisplatin inhibited tumor growth more than either drug alone. Further,the mice did not lose weight over the course of that study. (Data notshown.)

The tumor volume of each group of mice on day 42 was analyzed by one-wayANOVA followed by Tukey's test. The results of that analysis are shownin Table 8.

TABLE 8 Tumor volume analysis on day 42 Tumor p Value Mean Tumor growthcompare volume inhibition to control mm³ (±SD) (%) (Tukey's test) Salinevehicle 1339 (±411)  — — FGFR1-ECD.339-Fc 807 (±431) 39 <0.01 Cisplatin682 (±195) 49 <0.001 FGFR1-ECD.339-Fc + 382 (±210) 71 <0.001 Cisplatin

In order to determine whether combination of FGFR1-ECD.339-Fc andcisplatin resulted in enhanced (additive or synergistic) or decreased(antagonistic) antitumor activity, fractional tumor volume was analyzedas described in Example 1. The results of that analysis are shown inTable 9.

TABLE 9 Analysis of fractional tumor volume^(a) on day 42 relative tocontrol FGFR1- Ex- Ob- Expected/ ECD.339-Fc Cisplatin pected^(b) servedObserved^(c) FGFR1- 0.60 0.5 0.30 0.28 1.07 ECD.339-Fc + cisplatin^(a)Fractional tumor volume (FTV) = (Mean tumor volume (TV)treated)/(Mean TV control) ^(b)Expected = (FTV drug 1) × (FTV drug 2)^(c)Ratio of expected over observed, >2 = synergistic; ~1 = additive;<0.5 = antagonistic.

Those results show that the combination of FGFR1-ECD.339-Fc andcisplatin resulted in additive inhibition of tumor growth in thatexperiment.

B. FGFR1-ECD.339-Fc and Paclitaxel

The combination of FGFR1-ECD.339-Fc and paclitaxel was tested in theA549 human non-small cell lung cancer xenograft model, described above.FGFR1-ECD.339-Fc was formulated in 0.9% Saline for Injection USP at 3mg/ml. Paclitaxel was purchased from Bedford Laboratories (Bedford,Ohio; Cat. No. 1075029) and was formulated in 0.9% saline for injectioncontaining 5% dextrose at 3.6 mg/ml for a dose of 18 mg/kg.FGFR1-ECD.339-Fc was administered intraperitoneally (i.p.) at 15 mg/kgtwice per week for 5 weeks. Paclitaxel was administered i.p. at 18 mg/kgon days 8, 12, and 15.

When the average tumor volume in vehicle control group reached ˜500 mm³,the mice were euthanized by isoflurane inhalation and cervicaldislocation.

The mean tumor volume throughout the study for each set of mice is shownin FIG. 6. In that experiment, the combination of FGFR1-ECD.339-Fc andpaclitaxel inhibited tumor growth more than either drug alone. Further,the mice did not lose weight over the course of that study. (Data notshown.)

In order to determine whether the combination of FGFR1-ECD.339-Fc andpaclitaxel resulted in additive, synergistic, or antagonistic activity,fractional tumor volume on day 31 and day 38 was analyzed as describedin Example 1. The results of that analysis are shown in Table 10.

TABLE 10 Analysis of fractional tumor volume^(a) on day 31 and day 38FGFR1- Expected/ Day ECD.339-Fc Paclitaxel Expected^(b) ObservedObserved^(c) 31 0.84 0.57 0.48 0.26 1.81 38 0.89 0.50 0.45 0.39 1.13^(a)Fractional tumor volume (FTV) = (Mean tumor volume (TV)treated)/(Mean TV control) ^(b)Expected = (FTV drug 1) × (FTV drug 2)^(c)Ratio of expected over observed, >2 = synergistic; ~1 = additive;<0.5 = antagonistic.

Those results show that administration of FGFR1-ECD.339-Fc andpaclitaxel resulted in additive inhibition of tumor growth.

C. FGFR1-ECD.339-Fc and 5-FU

The combination of FGFR1-ECD.339-Fc and 5-fluorouracil (5-FU) was testedin the A549 human non-small cell lung cancer xenograft model, describedabove. FGFR1-ECD.339-Fc was formulated in 0.9% Saline for Injection USPat 3 mg/ml. 5-FU was purchased from Sigma-Aldrich (St. Louis, Mo.; Cat.No. F6627) and was initially dissolved in dimethyl sulfoxide (DMSO,Sigma-Aldrich, St. Louis, Mo.; Cat. No. D8418-50) at the concentrationof 50 mg/ml as a stock solution. The stock solution was further dilutedin 0.9% Sodium Chloride Injection USP to 6.6 mg/ml (for 33 mg/kgdosing). FGFR1-ECD.339-Fc was administered intraperitoneally (i.p.) at15 mg/kg twice per week for four weeks. 5-FU was administered i.p. at 33mg/kg twice a week for three weeks.

Mice were euthanized on day 31 by isoflurane inhalation and cervicaldislocation.

The mean tumor volume throughout the study for each set of mice is shownin FIG. 7. In that experiment, the combination of FGFR1-ECD.339-Fc and5-FU inhibited tumor growth more than either drug alone. Further, themice did not lose weight over the course of that study. (Data notshown.)

In order to determine whether the combination of FGFR1-ECD.339-Fc and5-FU resulted in additive, synergistic, or antagonistic activity,fractional tumor volume on day 31 was analyzed as described inExample 1. The results of that analysis are shown in Table 11.

TABLE 11 Analysis of fractional tumor volume^(a) on day 31 FGFR1-Expected/ Day ECD.339-Fc 5-FU Expected^(b) Observed Observed^(c) 31 0.841.04 0.89 0.38 2.33 ^(a)Fractional tumor volume (FTV) = (Mean tumorvolume (TV) treated)/(Mean TV control) ^(b)Expected = (FTV drug 1) ×(FTV drug 2) ^(c)Ratio of expected over observed, >2 = synergistic; ~1 =additive; <0.5 = antagonistic.

Those results show that administration of FGFR1-ECD.339-Fc and 5-FUresulted in synergistic inhibition of tumor growth in that experiment.The inventors note that similar experiments using 20 mg/kg or 50 mg/kg5-FU did not result in synergy. (Data not shown.)

D. FGFR1-ECD.339-Fc and Docetaxel

The combination of FGFR1-ECD.339-Fc and docetaxel was tested in the A549human non-small cell lung cancer xenograft model, described above.FGFR1-ECD.339-Fc was formulated in PBS at 3 mg/ml and administeredintraperitoneally (i.p.) at 15 mg/kg (300 μg/100 μl/mouse) twice a weekfor four weeks. Docetaxel was administered i.p. at 3 mg/kg or 10 mg/kgtwice per week for four weeks.

Mice in the albumin control group were euthanized on day 32 byisoflurane inhalation and cervical dislocation when the average tumorvolume in the group reached 1000 mm³.

The mean tumor volume throughout the study for each set of mice is shownin FIGS. 8A (3 mg/kg docetaxel) and 8B (10 mg/kg docetaxel). In thatexperiment, the combination of FGFR1-ECD.339-Fc and docetaxel inhibitedtumor growth more than either drug alone, at either dosage of docetaxel.Further, the mice did not lose weight over the course of that study, ateither dosage of docetaxel. (Data not shown.)

In order to determine whether the combination of FGFR1-ECD.339-Fc anddocetaxel resulted in additive, synergistic, or antagonistic activity,fractional tumor volume on day 32 was analyzed as described inExample 1. The results of that analysis are shown in Table 12.

TABLE 12 Analysis of fractional tumor volume^(a) on day 32 FGFR1-ECD.339- Ob- Expected/ Day Fc Docetaxel Expected^(b) served Observed^(c)32 (3 mg/kg 0.54 0.79 0.43 0.44 0.95 docetaxel) 32 (10 mg/kg 0.54 0.580.31 0.42 0.73 docetaxel) ^(a)Fractional tumor volume (FTV) = (Meantumor volume (TV) treated)/(Mean TV control) ^(b)Expected = (FTV drug 1)× (FTV drug 2) ^(c)Ratio of expected over observed, >2 = synergistic; ~1= additive; <0.5 = antagonistic.

Those results show that administration of FGFR1-ECD.339-Fc and docetaxelresulted in additive inhibition of tumor growth in that experiment.

E. FGFR1-ECD.339-Fc and Vincristine

The combination of FGFR1-ECD.339-Fc and vincristine was tested in theA549 human non-small cell lung cancer xenograft model, described above.FGFR1-ECD.339-Fc was formulated in 0.9% Saline for Injection USP at 1.5mg/ml for administration at 15 mg/kg (300 μg/200 μl per mouse), or 2mg/ml for administration at 20 mg/kg (400 μg/200 μl per mouse).Vincristine was obtained from Fluka-Sigma (St. Louis, Mo. 63103, Catalog#V8879) and was formulated in 0.9% Saline for Injection USP at 0.1 mg/mlor 0.15 mg/mL for administration at 1 mg/kg (0.02 μg/100 μl per mouse)or 1.5 mg/kg (0.03 μg/200 μl per mouse), respectively.

In the first experiment, FGFR1-ECD.339-Fc was administeredintraperitoneally (i.p.) at 15 mg/kg twice per week starting on day 1for six weeks, and vincristine was administered i.p. at 1 mg/kg on days8, 15, and 22. In the second experiment, FGFR1-ECD.339-Fc wasadministered intraperitoneally (i.p.) at 20 mg/kg twice per weekstarting on day 19 for seven weeks, and vincristine was administeredi.p. at 1.5 mg/kg on days 27, 34, and 41.

Mice from the first experiment were euthanized 46 days post tumorimplantation. In the second study, mice in the albumin control group andmice in the FGFR1-ECD.339-Fc group were euthanized 70 days post tumorimplantation, while mice in the vincristine-treated groups wereeuthanized 77 days post tumor implantation. All of the mice wereeuthanized by isoflurane inhalation and cervical dislocation.

The mean tumor volume throughout the study for each set of mice is shownin FIGS. 9A (1 mg/kg vincristine; dosing begun at day 1) and 9B (1.5mg/kg vincristine; dosing begun at day 19). In that experiment, thecombination of FGFR1-ECD.339-Fc and vincristine inhibited tumor growthmore than either drug alone, at either dosage of vincristine, and ateither dosing schedule. Further, at the lower dose of vincristine, themice did not lose weight. (Data not shown.) At the higher dose ofvincristine, the mice lost weight, indicating that the higher dose iscloser to the maximum tolerated dose. See FIG. 9C.

In order to determine whether the combination of FGFR1-ECD.339-Fc andvincristine resulted in additive, synergistic, or antagonistic activity,fractional tumor volume on days 39 and 46 for the first experiment, andon days 70 for the second experiment, was analyzed as described inExample 1. The results of that analysis are shown in Table 13.

TABLE 13 Analysis of fractional tumor volume^(a) Dose of FGFR1- FGFR1-Dose of ECD.339- Expected/ ECD.339-Fc Vincristine Day Fc VincristineExp.^(b) Obs. Observed^(c) 15 mg/kg 1 mg/kg 39 0.50 0.53 0.27 0.23 1.16(start day 1) 15 mg/kg 1 mg/kg 46 0.40 0.55 0.22 0.24 0.91 (start day 1)20 mg/kg 1.5 mg/kg   70 0.71 0.41 0.29 0.14 2.07 (start day 19)^(a)Fractional tumor volume (FTV) = (Mean tumor volume (TV)treated)/(Mean TV control) ^(b)Expected = (FTV drug 1) × (FTV drug 2)^(c)Ratio of expected over observed, >2 = synergistic; ~1 = additive;<0.5 = antagonistic.

Those results show that administration of FGFR1-ECD.339-Fc andvincristine resulted in additive inhibition of tumor growth at the lowerdose of vincristine, and synergistic inhibition of tumor growth at thehigher dose of vincristine.

F. FGFR1-ECD.339-Fc, Carboplatin, and Paclitaxel

The combination of FGFR1-ECD.339-Fc, carboplatin, and paclitaxel wastested in the A549 human non-small cell lung cancer xenograft model,described above. FGFR1-ECD.339-Fc was formulated in 0.9% Saline forInjection USP at 3 mg/ml (for administration at 15 mg/kg). Carboplatinwas obtained from Sigma-Aldrich (St. Luis, Mo. 63103, Catalog # C2538)and was formulated in 0.9% Saline for Injection USP at 2.5 mg/mL foradministration at 25 mg/kg (500 μg/200 μL per mouse). Paclitaxel wasobtained from LC Laboratories (Woburn, Mass. 01801; Catalog # P-9600)and was formulated in a solution of 50.3% Cremophor® and 49.7%dehydrated alcohol at 20 mg/mL as stock solution. The stock solution wasfurther diluted in 5% dextrose in 0.9% Saline for Injection USP at 3mg/mL for administration at 30 mg/kg (600 μg/200 μL per mouse).

FGFR1-ECD.339-Fc was administered intraperitoneally (i.p.) at 15 mg/kgtwice per week starting on day 7 for three weeks. Carboplatin wasadministered i.p. at 25 mg/kg twice per week starting on day 7 for threeweeks. Paclitaxel was administered i.p. at 30 mg/kg twice per weekstarting on day 8 for three weeks.

Mice were euthanized on day 34 in single agent groups and on day 41 incombination groups. Mice were euthanized by isoflurane inhalation andcervical dislocation.

The mean tumor volume throughout the study for each set of mice is shownin FIG. 10. In that experiment, the combination of FGFR1-ECD.339-Fc,carboplatin, and paclitaxel inhibited tumor growth more than any of thedrugs alone, and also more than the combination of carboplatin andpaclitaxel. No weight loss was observed during the course of that study.(Data not shown.)

In order to determine whether the combination of FGFR1-ECD.339-Fc,carboplatin, and paclitaxel resulted in additive, synergistic, orantagonistic activity, fractional tumor volume on day 28 was analyzed asdescribed in Example 1. The results of that analysis are shown in Table14.

TABLE 14 Analysis of fractional tumor volume^(a) FGFR1- ECD.339-Paclitaxel + Expected/ Day Fc carboplatin Expected^(b) ObservedObserved^(c) 28 0.58 0.27 0.16 0.14 1.15 ^(a)Fractional tumor volume(FTV) = (Mean tumor volume (TV) treated)/(Mean TV control) ^(b)Expected= (FTV drug 1) × (FTV drug 2) ^(c)Ratio of expected over observed, >2 =synergistic; ~1 = additive; <0.5 = antagonistic.

Those results show that administration of FGFR1-ECD.339-Fc, carboplatin,and paclitaxel resulted in additive inhibition of tumor growth in thatexperiment.

Example 4: Administration of FGFR1-ECD.339-Fc in Combination withVarious Chemotherapeutics in the Colo205 Colon Cancer Xenograft Model

Colo205 cells were purchased from ATCC (Manassas, Va.; Cat. No. CCL-222)and were cultured for 3 passages in RPMI1640 media, 10% FBS, and 1%L-Glutamine at 37° C. in a humidified atmosphere with 5% CO₂. Cells wereresuspended in a solution of 50%/v PBS and 50%/v Matrigel at aconcentration of 25 million cells/ml. Resuspended cells were kept on iceuntil implantation. Six weeks old female SCID mice were purchased fromCharles River Laboratories (Wilmington, Mass.) and were acclimated for 1week before the start of the study. On day 0, 2.5 million cells/100 μlwere implanted over the right flank of the each mouse using a 27G1/2needle. One day after tumor implantation, the mice were randomizedaccording to the body weight.

The tumor volume and body weight of the mice were monitored twice a weekthroughout each study. The tumor volume was measured and calculatedusing the method and formula described in Example 1.

The tumor volume of each group of mice at the end of each study wasanalyzed by one-way ANOVA followed by Tukey's test. Fractional tumorvolume analysis was then used to assess the degree of enhanced (additiveor synergistic) or decreased (antagonistic) tumor growth inhibitionachieved following administration of FGFR1-ECD.339-Fc with one or moreadditional chemotherapeutic molecules.

Certain study details, and the results for each combination, arediscussed below.

A. FGFR1-ECD.339-Fc, 5-FU, Leucovorin, and Bevacizumab

FGFR1-ECD.339-Fc was formulated in 0.9% Sodium Chloride Injection USP(Henry Schein, Inc, Melville, N.Y.; Cat. No. 1533826) at 2 mg/ml andstored in screw cap microcentrifuge tubes at −80° C. The negativecontrol reagent, human albumin, was purchased from Grifols USA (LosAngeles, Calif.; Cat. No. NDC 61953-0002-1) and was formulated in PBS at3 mg/ml. 5-FU was purchased from Sigma-Aldrich (St. Louis, Mo.; Cat. No.F6627) and was initially dissolved in dimethyl sulfoxide (DMSO,Sigma-Aldrich, St. Louis, Mo.; Cat. No. D8418-50) at the concentrationof 50 mg/ml as a stock solution. The stock solution was further dilutedin 0.9% Sodium Chloride Injection USP to 2 mg/ml (for 10 mg/kg dosing),to 4 mg/ml (for 20 mg/kg dosing), and to 6 mg/ml (for 30 mg/kg dosing).Leucovorin (LV) was also purchased from Sigma-Aldrich (Cat. No. F8259)and was formulated in 0.9% Sodium Chloride Injection USP at 2 mg/ml (for10 mg/kg dosing), to 4 mg/ml (for 20 mg/kg dosing), and to 6 mg/ml (for30 mg/kg dosing). Bevacizumab was purchased from Genentech, Inc (SouthSan Francisco, Calif.; Cat. No. 15734) and was diluted in 0.9% SodiumChloride Injection USP to 0.2 mg/ml (for 1 mg/kg dosing).

In the first experiment, FGFR1-ECD.339-Fc was combined with threedifferent concentrations of 5-FU/leucovorin. In the second experiment,FGFR1-ECD.339-Fc was combined with either bevacizumab or5-FU/leucovorin, or both. The grouping and dosing schedule for theexperiments are shown in Table 15. The double line separates the groupsfrom the two experiments.

TABLE 15 Dosing Schedule (Dosing started one day post tumorimplantation) Drug 1 Drug 2 Drug 3 (Dose, route, (Dose, route, (Dose,route, N Treatment Schedule) Schedule) Schedule) 10 Albumin + Albumin(10 mg/kg, Saline (100 μl, — Vehicle i.p., 3x/wk × q.d. × 5 days) 4 wks)10 FGFR1- FGFR1- — — ECD.339-Fc ECD.339-Fc (15 mg/kg, i.p., 3x/wk × 4wks) 10 5-Fu/LV 5-FU (10 mg/kg, LV (10 mg/kg, i.p., q.d. × 5 days) i.p.,q.d. × 5 days) FGFR1- FGFR1- 5-FU (10 mg/kg, LV (10 mg/kg, ECD.339-Fc +ECD.339-Fc (15 mg/kg, i.p., q.d. × 5 days) i.p., q.d. × 5 5-Fu/LV i.p.,3x/wk × days) (10 mg/kg) 4 wks) 10 5-Fu/LV 5-FU (20 mg/kg, LV (20 mg/kg,i.p., q.d. × 5 days) i.p., q.d. × 5 days) FGFR1- FGFR1- 5-FU (20 mg/kg,LV (20 mg/kg, ECD.339-Fc + ECD.339-Fc (15 mg/kg, i.p., q.d. × 5 days)i.p., q.d. × 5 5-Fu/LV i.p., 3x/wk × days) (20 mg/kg) 4 wks) 10 5-Fu/LV5-FU (30 mg/kg, LV (30 mg/kg, i.p., q.d. × 5 days) i.p., q.d. × 5 days)FGFR1- FGFR1- 5-FU (30 mg/kg, LV (30 mg/kg, ECD.339-Fc + ECD.339-Fc (15mg/kg, i.p., q.d. × 5 days) i.p., q.d. × 5 5-Fu/LV i.p., 3x/wk × days)(30 mg/kg) 4 wks) 10 Bevacizumab Bev (1 mg/kg, — i.p., 2x/wk × 4 wks) 10Bevacizumab + Bev (1 mg/kg, 5-Fu/LV (10 mg/kg — 5-Fu/LV i.p., 2x/wk × 4wks) each, i.p., q.d. × 5 days) 10 FGFR1- FGFR1- — — ECD.339-FcECD.339-Fc (15 mg/kg, i.p., 3x/wk × 4 wks) 10 FGFR1- FGFR1- Bev (1mg/kg, — ECD.339-Fc + ECD.339-Fc (15 mg/kg, i.p., 2x/wk × 4 wks)Bevacizumab i.p., 3x/wk × 4 wks) 10 FGFR1- FGFR1- Bev (1 mg/kg, 5-Fu/LV(10 mg/kg ECD.339-Fc + ECD.339-Fc (15 mg/kg, i.p., 2x/wk × 4 wks) each,Bevacizumab + i.p., 3x/wk × i.p., q.d. × 5 5-Fu/LV 4 wks) days)

When the average tumor volume in a group was near 600 mm³, the mice inthat group were euthanized by isoflurane inhalation and cervicaldislocation. The mean tumor volume throughout the study for each set ofmice is shown in FIG. 11. At 20 mg/kg 5-FU/leucovorin and 30 mg/kg5-FU/leucovorin, the mice lost about 3 grams and about 4 grams of bodyweight, respectively (about 14% and 19%, respectively). (Data notshown.) No weight loss in the mice was observed in any of the remaininggroups. (Data not shown.)

The order of effectiveness of the various treatments was evaluated twoways: 1) the average tumor volume in each group on a single time point(day); and 2) time for the average tumor volume in each group to reach500 mm³ (see the dotted line in FIG. 11E).

The results of that experiment according to both methods showed that theantitumor effect of various treatment groups was in the following order:FGFR1-ECD.339-Fc, bevacizumab, and 5-Fu/leucovorin>FGFR1-ECD.339-Fc andbevacizumab>bevacizumab and5-Fu/leucovorin=bevacizumab>FGFR1-ECD.339-Fc>vehicle.

The average tumor volume of each group of mice in the second experimenton day 24 was analyzed by one-way ANOVA followed by Tukey's test. Theresults of that analysis are shown in Table 16.

TABLE 16 Tumor volume analysis on day 24 Mean Tumor p Value Tumor growthcompare volume inhibition to control mm³ (±SD) (%) (Tukey's test)Albumin + vehicle 693 (±159) — — FGFR1-ECD.339-Fc 509 (±144) 26 <0.055-FU/LV 511 (±196) 26 >0.05 Bevacizumab 405 (±56)  41 <0.001Bevacizumab + 399 (±131) 42 <0.001 5-Fu/LV FGFR1-ECD.339-Fc + 322 (±116)53 <0.001 Bevacizumab FGFR1-ECD.339-Fc + 269 (±83)  61 <0.001Bevacizumab + 5-Fu/LV

In order to determine whether administration of FGFR1-ECD.339-Fc and5-FU/leucovorin at various concentrations; FGFR1-ECD.339-Fc andbevacizumab; or FGFR1-ECD.339-Fc, bevacizumab, and 5-Fu/leucovorinresulted in additive, synergistic, or antagonistic activity, fractionaltumor volume was analyzed as described in Example 1. The results of thatanalysis are shown in Table 17.

TABLE 17 Analysis of fractional tumor volume^(a) FGFR1- ECD. Ex- Ob-Expected/ 339-Fc pected^(b) served Observed^(c) 5-FU/LV (10 mg/kg) Day16 0.76 0.86 0.67 0.62 1.08 5-FU/LV (20 mg/kg) Day 16 0.76 0.56 0.420.42 1.00 5-FU/LV (30 mg/kg) Day 16 0.76 0.36 0.27 0.28 0.96 BevacizumabDay 24 0.73 0.58 0.42 0.46 0.91 Bevacizumab/ 5-Fu/LV Day 24 0.73 0.570.42 0.38 1.10 ^(a)Fractional tumor volume (FTV) = (Mean tumor volume(TV) treated)/(Mean TV control) ^(b)Expected = (FTV drug 1) × (FTV drug2) ^(c)Ratio of expected over observed, >2 = synergistic; ~1 = additive;<0.5 = antagonistic.

Those results show that administration of FGFR1-ECD.339-Fc and5-FU/leucovorin at various concentrations; FGFR1-ECD.339-Fc andbevacizumab; or FGFR1-ECD.339-Fc, bevacizumab, and 5-Fu/leucovorin,resulted in additive inhibition of tumor growth.

B. FGFR1-ECD.339-Fc, 5-FU, Leucovorin, and Oxaliplatin

The combination of FGFR1-ECD.339-Fc, 5-FU, leucovorin, and oxaliplatinwas tested in the Colo205 human colon cancer xenograft model, describedabove. FGFR1-ECD.339-Fc was formulated in PBS at 3 mg/ml (foradministration at 15 mg/kg). 5-FU was purchased from Sigma-Aldrich (St.Louis, Mo.; Cat. No. F6627) and was initially dissolved in dimethylsulfoxide (DMSO, Sigma-Aldrich, St. Louis, Mo.; Cat. No. D8418-50) atthe concentration of 50 mg/ml as a stock solution. The stock solutionwas further diluted in 0.9% Sodium Chloride Injection USP to 2 mg/ml(for 10 mg/kg dosing). Leucovorin was also purchased from Sigma-Aldrich(Cat. No. F8259) and was formulated in 0.9% Sodium Chloride InjectionUSP at 2 mg/ml (for 10 mg/kg dosing). Oxaliplatin was obtained from LClaboratories (Woburn, Mass. 01801; Catalog #0-7111) and was formulatedin 5% Dextrose Injection (Baxter, Deerfield, Ill. 60015; Catalog#2B0082) at 1 mg/mL, 2 mg/mL, and 3 mg/ml for administration at 5 mg/kg(100 μg/100 μL per mouse), 10 mg/kg (200 μg/100 μL per mouse), and 15mg/kg (300 μg/100 μl per mouse).

FGFR1-ECD.339-Fc was administered intraperitoneally (i.p.) at 15 mg/kgtwice per week starting on day 1 for four weeks. 5-FU and leucovorinwere each administered i.p. at 10 mg/kg daily for five days. Oxaliplatinwas administered i.p. at 5 mg/kg, 10 mg/kg, or 15 mg/kg in one dose onday 1.

Mice were euthanized on day 28 by isoflurane inhalation and cervicaldislocation.

The mean tumor volume throughout the study for each set of mice is shownin FIG. 12. In that experiment, the combination of FGFR1-ECD.339-Fc,5-FU, leucovorin, and oxaliplatin inhibited tumor growth more thanFGFR1-ECD.339-Fc alone or the combination 5-FU, leucovorin, andoxaliplatin. Minimal weight loss in the mice was observed at 5 mg/kgoxaliplatin (0.78 grams, or 4% body weight); moderate weight loss wasobserved at 10 mg/kg (2.6 grams, or 13% body weight); and more severeweight loss was observed at 15 mg/kg oxaliplatin twice per week (3.7grams, or 19% body weight). (Data not shown.)

In order to determine whether the combination of FGFR1-ECD.339-Fc, 5-FU,leucovorin, and oxaliplatin resulted in additive, synergistic, orantagonistic activity, fractional tumor volume on day 17 was analyzed asdescribed in Example 1. The results of that analysis are shown in Table18.

TABLE 18 Analysis of fractional tumor volume^(a) on day 17 FGFR1-Oxaliplatin ECD.339- Oxaliplatin/ Ob- Expected/ dose Fc 5-FU/LVExpected^(b) served Observed^(c)  5 mg/kg 0.77 0.78 0.60 0.47 1.27 10mg/kg 0.77 0.63 0.48 0.5 0.96 15 mg/kg 0.77 0.46 0.35 0.36 0.97 twiceper week ^(a)Fractional tumor volume (FTV) = (Mean tumor volume (TV)treated)/(Mean TV control) ^(b)Expected = (FTV drug 1) × (FTV drug 2)^(c)Ratio of expected over observed, >2 = synergistic; ~1 = additive;<0.5 = antagonistic.

Those results show that administration of FGFR1-ECD.339-Fc, 5-FU,leucovorin, and oxaliplatin resulted in additive inhibition of tumorgrowth at each dosage of oxaliplatin tested in that experiment.

Example 5: Administration of FGFR1-ECD.339-Fc, Doxorubicin, andPaclitaxel in the JIMT-1 Breast Cancer Xenograft Model

Six week old female SCID mice were purchased from Charles RiverLaboratories (Wilmington, Mass.) and were acclimated for 1 week beforethe start of the study. Human breast cancer cell line JIMT-1 was used asthe tumor model and was purchased from Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH (DMSZ, Braunschweig, Germany; Cat.No. ACC 589). The cells were cultured for ten passages in DMEM+10% FBS,1% L-glutamine, and 1% penicillin/streptomycin at 37° C. in a humidifiedatmosphere with 5% CO₂. When the cultured cells reached 85-90%confluence, cells were harvested and resuspended in cold Ca²⁺ and Mg²⁺free phosphate buffered saline (PBS) containing 50% Matrigel at 5×10⁷cells per milliliter. The cells were implanted subcutaneously over theright flank of the mice at 5×10⁶ cells/100 μl/mouse. One day after tumorimplantation, mice were randomized according to body weight at 10 miceper group.

FGFR1-ECD.339-Fc was formulated in 0.9% Saline for Injection USP at 3mg/ml and administered intraperitoneally (i.p.) at 15 mg/kg (300 μg/100μl/mouse) twice per week for five weeks beginning on day 7. Doxorubicinwas obtained from Sigma-Aldrich (St. Luis, Mo.; Catalog #44583) and wasformulated in 0.9% Saline for Injection USP at 0.05 mg/mL foradministration at 0.5 mg/kg (10 μg/200 μL per mouse) once per week forfive weeks, beginning on day 7. Paclitaxel was obtained from BedfordLaboratories (Bedford, Ohio; Cat. No. 1075029) and was formulated in asolution of 50.3% Cremophor® and 49.7% dehydrated alcohol. The stocksolution was diluted in 5% dextrose/0.9% Saline for Injection USP to afinal concentration of 3 mg/mL paclitaxel in 16.8% Cremophor®, 16.6%dehydrated alcohol, 3.3% dextrose, 0.6% Saline for Injection USP.Paclitaxel was administered at 30 mg/kg (60 μg/200 μL per mouse) twiceper week for five weeks beginning on day 7.

The tumor volume and body weight of the mice were monitored twice a weekthroughout the study. The tumor volume was measured and calculated usingthe method and formula described in Example 1.

Mice were euthanized on day 42 by isoflurane inhalation and cervicaldislocation.

The mean tumor volume throughout the study for each set of mice is shownin FIG. 13. In that experiment, the combination of FGFR1-ECD.339-Fc,doxorubicin, and paclitaxel inhibited tumor growth more thanFGFR1-ECD.339-Fc alone or the combination of doxorubicin and paclitaxelwithout FGFR1-ECD.339-Fc. No weight loss was observed over the course ofthe study. (Data not shown.)

Fractional tumor volume analysis was used to assess the degree ofenhanced (additive or synergistic) or decreased (antagonistic) tumorgrowth inhibition by the combination of FGFR1-ECD.339-Fc, doxorubicin,and paclitaxel. The results of that analysis are shown in Table 19.

TABLE 19 Analysis of fractional tumor volume^(a) on day 21 FGFR1-Doxorubicin ECD.339- and Expected/ Fc paclitaxel Expected^(b) ObservedObserved^(c) Day 21 0.93 0.59 0.55 0.50 1.10 ^(a)Fractional tumor volume(FTV) = (Mean tumor volume (TV) treated)/(Mean TV control) ^(b)Expected= (FTV drug 1) × (FTV drug 2) ^(c)Ratio of expected over observed, >2 =synergistic; ~1 = additive; <0.5 = antagonistic.

Those results demonstrate that the combination of FGFR1-ECD.339-Fc,doxorubicin, and paclitaxel resulted in additive inhibition of tumorgrowth in that experiment.

Example 6: Treatment with FGFR1-ECD.339-Fc and KDR ECD-Fc in Mice Having11520 Lung Xenograft Tumor Cells Showed Tumor Inhibition

SCID CB17 mice at 8 weeks of age were administered either vehicle,kinase insert domain receptor (KDR) cDNA (Five Prime Therapeutics, SouthSan Francisco, Calif.) (which expresses a protein that acts as a VEGFantagonist and VEGF trap), FGFR1 cDNA (Five Prime Therapeutics, SouthSan Francisco, Calif.), or a combination of KDR and FGFR1, byhydrodynamic tail vein transfection (TVT), substantially as described inChen et al., Human Gene Therapy 16(1): 126-131 (2005), and theninoculated 4 days later with 5×10⁶ H520 lung xenograft tumor cells s.c.on the flank, with the cells in a 1:1 mixture of Matrigel in 100μ1 totalvolume. KDR and FGFR1 cDNA constructs each contain their respectiveextracellular domain fused to an IgG1 Fc domain. Tumor volumemeasurements were made at approximately 10 day intervals. On day 29, thetumor volume was reduced in groups treated with single agents relativeto vehicle (Mann Whitney test P<0.05). On day 29, the tumor volume wasreduced in the combination group relative to groups treated with singleagents (Mann Whitney test P<0.001).

Example 7: Administration of FGFR1-ECD.339-Fc and Cisplatin/Etoposide inthe DMS 53 Small Cell Lung Cancer (SCLC) Xenograft Model

Six week old female SCID mice were purchased from Charles RiverLaboratories (Wilmington, Mass.) and were acclimated for 1 week beforethe start of the study. Human small cell lung cancer (SCLC) cell lineDMS 53 was used as the tumor model and was purchased from ATCC(Manassas, Va.; Cat. No. CRL-2062). The cells were cultured for threepassages in Waymouth's MB 752/1 medium+10% FBS+2 mM L-glutamine at 37°C. in a humidified atmosphere with 5% CO₂. When the cultured cellsreached 85-90% confluence, cells were harvested and resuspended in coldCa²⁺ and Mg²⁺ free phosphate buffered saline (PBS) containing 50%Matrigel at 5×10⁷ cells per milliliter. The cells were implantedsubcutaneously over the right flank of the mice at 5×10⁶ cells/100μl/mouse. After tumors reached a size of 100-125 mm³, mice were sortedand randomized so each group (n=10) has the approximately the sameaverage tumor volume, and treatment initiated according to Table 20below.

FGFR1-ECD.339-Fc was formulated in PBS at 3 mg/ml and administeredintraperitoneally (i.p.) at 15 mg/kg (300 μg/100 μl/mouse) twice a weekfor four weeks. Cisplatin was purchased from Amatheon, Inc., Miami, Fla.(Cat No. 5539-0112-50) pre-formulated at concentration of 1 mg/ml. 0.45ml of cisplating stock solution (1 mg/ml) was added to 1.05 ml of 5%dextrose solution, for a 1.5 ml working solution with a concentration of0.3 mg/ml. Each mouse received 100 μl of working solution (0.3 mg/ml) toprovide a dose of 3 mg/kg every 7 or 21 days depending on group.Etoposide was purchased from Amatheon, Inc (Cat. No. 5539-0291-01)pre-formulated at concentration of 20 mg/ml. 0.140 ml of stock solution(20 mg/ml) was added to 3.36 ml of 5% dextrose solution, for a 3.5 mlworking solution with a concentration of 0.8 mg/ml. Each mouse received50 μl of working solution (0.8 mg/ml) to provide a dose of 4 mg/kg for 3days in a row. Etoposide dosing was repeated every 7 or 21 daysdepending on group. In combination groups, FGFR1-ECD.339-Fc andcisplatin/etoposide was administered concurrently. Human albumin waspurchased from Grifols USA (Los Angeles, Calif.; Cat. No. NDC61953-0002-1), diluted to a working stock (3 mg/ml) with 0.9% sodiumchloride, and was used as negative control at 300 μg/100 μl/mouse (15mg/kg). The dosing schedule for each set of mice is shown in Table 20.

TABLE 20 FGFR1- Cisplatin Etoposide ECD.339- (mg/kg, (mg/kg, Fc (mg/kg,Group Drugs schedule) schedule) schedule) Route 1 Albumin 15 mg/kg 0 0 0IP 2 FGFR1-ECD.339-Fc 0 0 15, IP 2x/week 3 Cisplatin/Etoposide 3, onceevery 4, qd × 3 0 IP 21 days every 21 days 4 Cisplatin/Etoposide/ 3,once every 4, qd × 3 15, IP FGFR1-ECD.339-Fc 21 days every 21 2x/weekdays 5 Cisplatin/Etoposide 3, once every 4, qd × 3 0 IP 7 days every 7days 6 Cisplatin/Etoposide/ 3, once every 4, qd × 3 15, IPFGFR1-ECD.339-Fc 7 days every 7 days 2x/week

Example 8: Administration of FGFR1-ECD.339-Fc and Topotecan in the DMS53 Small Cell Lung Cancer (SCLC) Xenograft Model

Six week old female SCID mice were purchased from Charles RiverLaboratories (Wilmington, Mass.) and were acclimated for 1 week beforethe start of the study. Human small cell lung cancer (SCLC) cell lineDMS 53 was used as the tumor model and was purchased from ATCC(Manassas, Va.; Cat. No. CRL-2062). The cells were cultured for threepassages in Waymouth's MB 752/1 medium+10% FBS+2 mM L-glutamine at 37°C. in a humidified atmosphere with 5% CO₂. When the cultured cellsreached 85-90% confluence, cells were harvested and resuspended in coldCa²⁺ and Mg²⁺ free phosphate buffered saline (PBS) containing 50%Matrigel at 5×10⁷ cells per milliliter. The cells were implantedsubcutaneously over the right flank of the mice at 5×10⁶ cells/100μl/mouse. After tumors reached a size of 100-125 mm³, mice were sortedand randomized so each group (n=10) has the approximately the sameaverage tumor volume, and treatment initiated according to Table 21below.

FGFR1-ECD.339-Fc was formulated in PBS at 3 mg/ml and administeredintraperitoneally (i.p.) at 15 mg/kg (300 μg/100 μl/mouse) twice a weekfor four weeks. Topotecan powder was purchased from Sigma-Aldrich, Inc.(Cat No. T2705-50MG) and a 5 mg/ml stock solution made in a 5% dextrosesolution. Dilutions are also made with 5% dextrose solution. 0.6 ml ofstock solution (5 mg/ml) was added to 5.4 ml of 5% dextrose solution,for a 6 ml working solution with a concentration of 0.5 mg/ml. Eachmouse received 100 μl of working solution (0.5 mg/ml) to give a dose of2.5 mg/kg. Topotecan dosing was repeated every 7 or 21 days depending ongroup. In combination groups, FGFR1-ECD.339-Fc and topotecan wasadministered concurrently. Human albumin was purchased from Grifols USA(Los Angeles, Calif.; Cat. No. NDC 61953-0002-1), diluted to a workingstock (3 mg/ml) with 0.9% sodium chloride, and was used as negativecontrol at 300 μg/100 μl/mouse (15 mg/kg). The dosing schedule for eachset of mice is shown in Table 21.

TABLE 21 FGFR1- ECD.339- Topotecan Fc (mg/kg, (mg/kg, Group Drugsschedule) schedule) Route 1 Albumin 15 mg/kg 0 0 IP 2 FGFR1-ECD.339-Fc 015, IP 2x/week 3 Topotecan 2.5, qd × 5 0 IP every 21 days 4Topotecan/FGFR1- 2.5, qd × 5 15, IP ECD.339-Fc every 21 2x/week days 5Topotecan 2.5, qd × 5 0 IP every 7 days 6 Topotecan/FGFR1- 2.5, qd × 515, IP ECD.339-Fc every 7 days 2x/week

TABLE OF SEQUENCES

Table 22 lists certain sequences discussed herein. FGFR1 sequences areshown without the signal peptide, unless otherwise indicated.

TABLE 22 Sequences and Descriptions SEQ ID NO Description Sequence  1Full-length human MWSWKCLLFW AVLVTATLCT ARPSPTLPEQ AQPWGAPVEVFGFR1 ECD (with ESFLVHPGDL LQLRCRLRDD VQSINWLRDG VQLAESNRTRsignal peptide); ITGEEVEVQD SVPADSGLYA CVTSSPSGSD TTYFSVNVSDSP-hFGFR1-ECD.353 ALPSSEDDDD DDDSSSEEKE TDNTKPNPVA PYWTSPEKMEKKLHAVPAAK TVKFKCPSSG TPNPTLRWLK NGKEFKPDHRIGGYKVRYAT WSIIMDSVVP SDKGNYTCIV ENEYGSINHTYQLDVVERSP HRPILQAGLP ANKTVALGSN VEFMCKVYSDPQPHIQWLKH IEVNGSKIGP DNLPYVQILK TAGVNTTDKEMEVLHLRNVS FEDAGEYTCL AGNSIGLSHH SAWLTVLEAL EERPAVMTSP LYLE  2Full-length human RPSPTLPEQ AQPWGAPVEV ESFLVHPGDL LQLRCRLRDDFGFR1 ECD (without VQSINWLRDG VQLAESNRTR ITGEEVEVQD SVPADSGLYAsignal peptide);  CVTSSPSGSD TTYFSVNVSD ALPSSEDDDD DDDSSSEEKEhFGFR1-ECD.353 TDNTKPNPVA PYWTSPEKME KKLHAVPAAK TVKFKCPSSGTPNPTLRWLK NGKEFKPDHR IGGYKVRYAT WSIIMDSVVPSDKGNYTCIV ENEYGSINHT YQLDVVERSP HRPILQAGLPANKTVALGSN VEFMCKVYSD PQPHIQWLKH IEVNGSKIGPDNLPYVQILK TAGVNTTDKE MEVLHLRNVS FEDAGEYTCLAGNSIGLSHH SAWLTVLEAL EERPAVMTSP LYLE  3 SP-hFGFR1-ECD.339MWSWKCLLFW AVLVTATLCT ARPSPTLPEQ AQPWGAPVEVESFLVHPGDL LQLRCRLRDD VQSINWLRDG VQLAESNRTRITGEEVEVQD SVPADSGLYA CVTSSPSGSD TTYFSVNVSDALPSSEDDDD DDDSSSEEKE TDNTKPNPVA PYWTSPEKMEKKLHAVPAAK TVKFKCPSSG TPNPTLRWLK NGKEFKPDHRIGGYKVRYAT WSIIMDSVVP SDKGNYTCIV ENEYGSINHTYQLDVVERSP HRPILQAGLP ANKTVALGSN VEFMCKVYSDPQPHIQWLKH IEVNGSKIGP DNLPYVQILK TAGVNTTDKEMEVLHLRNVS FEDAGEYTCL AGNSIGLSHH SAWLTVLEAL  4 hFGFR1-ECD.339RPSPTLPEQ AQPWGAPVEV ESFLVHPGDL LQLRCRLRDDVQSINWLRDG VQLAESNRTR ITGEEVEVQD SVPADSGLYACVTSSPSGSD TTYFSVNVSD ALPSSEDDDD DDDSSSEEKETDNTKPNPVA PYWTSPEKME KKLHAVPAAK TVKFKCPSSGTPNPTLRWLK NGKEFKPDHR IGGYKVRYAT WSIIMDSVVPSDKGNYTCIV ENEYGSINHT YQLDVVERSP HRPILQAGLPANKTVALGSN VEFMCKVYSD PQPHIQWLKH IEVNGSKIGPDNLPYVQILK TAGVNTTDKE MEVLHLRNVS FEDAGEYTCL AGNSIGLSHH SAWLTVLEAL  5SP-hFGFR1-ECD.339- MWSWKCLLFW AVLVTATLCT ARPSPTLPEQ AQPWGAPVEV FcESFLVHPGDL LQLRCRLRDD VQSINWLRDG VQLAESNRTRITGEEVEVQD SVPADSGLYA CVTSSPSGSD TTYFSVNVSDALPSSEDDDD DDDSSSEEKE TDNTKPNPVA PYWTSPEKMEKKLHAVPAAK TVKFKCPSSG TPNPTLRWLK NGKEFKPDHRIGGYKVRYAT WSIIMDSVVP SDKGNYTCIV ENEYGSINHTYQLDVVERSP HRPILQAGLP ANKTVALGSN VEFMCKVYSDPQPHIQWLKH IEVNGSKIGP DNLPYVQILK TAGVNTTDKEMEVLHLRNVS FEDAGEYTCL AGNSIGLSHH SAWLTVLEALEPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQYNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKSRWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK  6 hFGFR1-ECD.339-FcRPSPTLPEQ AQPWGAPVEV ESFLVHPGDL LQLRCRLRDDVQSINWLRDG VQLAESNRTR ITGEEVEVQD SVPADSGLYACVTSSPSGSD TTYFSVNVSD ALPSSEDDDD DDDSSSEEKETDNTKPNPVA PYWTSPEKME KKLHAVPAAK TVKFKCPSSGTPNPTLRWLK NGKEFKPDHR IGGYKVRYAT WSIIMDSVVPSDKGNYTCIV ENEYGSINHT YQLDVVERSP HRPILQAGLPANKTVALGSN VEFMCKVYSD PQPHIQWLKH IEVNGSKIGPDNLPYVQILK TAGVNTTDKE MEVLHLRNVS FEDAGEYTCLAGNSIGLSHH SAWLTVLEAL EPKSSDKTHT CPPCPAPELLGGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRDELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK  7hFGFR1 signal peptide MWSWKCLLFWAVLVTATLCTA  8 F Cc237SEPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQYNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKSRWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK  9 Exemplary Fc #1ERKCCVECPP CPAPPVAGPS VFLFPPKPKD TLMISRTPEVTCVVVDVSHE DPEVQFNWYV DGVEVHNAKT KPREEQFNSTFRVVSVLTVV HQDWLNGKEY KCKVSNKGLP APIEKTISKTKGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAVEWESNGQPEN NYKTTPPMLD SDGSFFLYSK LTVDKSRWQQGNVFSCSVMH EALHNHYTQK SLSLSPGK 10 Exemplary Fc #2ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK DTLMISRTPEVTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISKAKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIAVEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQEGNVFSCSVM HEALHNHYTQ KSLSLSLGK

1. A method of treating lung cancer in a human subject comprisingadministering to the subject an effective amount of a fibroblast growthfactor receptor 1 (FGFR1) extracellular domain (ECD) fusion molecule andat least one additional therapeutic agent selected from docetaxel,paclitaxel, vincristine, carboplatin, and topotecan, wherein the FGFR1ECD fusion molecule comprises an FGFR1 ECD comprising an amino acidsequence selected from SEQ ID NOs: 1 to 4 and a fusion partner.
 2. Themethod of claim 1, wherein the at least one additional therapeutic agentis docetaxel.
 3. The method of claim 1, wherein the at least oneadditional therapeutic agent is paclitaxel.
 4. (canceled)
 5. (canceled)6. The method of claim 1, wherein the at least one additionaltherapeutic agent is vincristine.
 7. (canceled)
 8. The method of claim1, wherein the at least one additional therapeutic agent is topotecan.9. The method of claim 1, wherein the method comprises administering tothe subject at least two additional therapeutic agents selected fromdocetaxel, paclitaxel, vincristine, carboplatin, and topotecan. 10.(canceled)
 11. The method of claim 9, wherein the at least twoadditional therapeutic agents are paclitaxel and carboplatin. 12.(canceled)
 13. (canceled)
 14. (canceled)
 15. (canceled)
 16. (canceled)17. (canceled)
 18. The method of claim 1, wherein the fusion partner isan Fc.
 19. The method of claim 1, wherein the FGFR1 ECD fusion moleculecomprises a sequence selected from SEQ ID NO: 5 and SEQ ID NO:
 6. 20.(canceled)
 21. The method of claim 1, wherein the lung cancer isnon-small cell lung cancer.
 22. The method of claim 2, wherein thefusion partner is an Fc.
 23. The method of claim 2, wherein the FGFR1ECD fusion molecule comprises a sequence selected from SEQ ID NO: 5 andSEQ ID NO:
 6. 24. The method of claim 2, wherein the cancer is non-smallcell lung cancer.
 25. The method of claim 9, wherein the fusion partneris an Fc.
 26. The method of claim 9, wherein the FGFR1 ECD fusionmolecule comprises a sequence selected from SEQ ID NO: 5 and SEQ ID NO:6.
 27. The method of claim 9, wherein the lung cancer is non-small celllung cancer.
 28. The method of claim 11, wherein the fusion partner isan Fc.
 29. The method of claim 11, wherein the FGFR1 ECD fusion moleculecomprises a sequence selected from SEQ ID NO: 5 and SEQ ID NO:
 6. 30.The method of claim 11, wherein the lung cancer is non-small cell lungcancer.